Disease therapy by inducing immune response to Trop-2 expressing cells

ABSTRACT

The present invention concerns compositions and methods of use of bispecific antibodies comprising at least one binding site for Trop-2 (EGP-1) and at least one binding site for CD3. The bispecific antibodies are of use for inducing an immune response against a Trop-2 expressing tumor, such as carcinoma of the esophagus, pancreas, lung, stomach, colon, rectum, urinary bladder, breast, ovary, uterus, kidney or prostate. The methods may comprising administering the bispecific antibody alone, or with one or more therapeutic agents such as antibody-drug conjugates, interferons (preferably interferon-α), and/or checkpoint inhibitor antibodies. The bispecific antibody is capable of targeting effector T cells, NK cells, monocytes or neutrophils to induce leukocyte-mediated cytotoxicity of Trop-2 +  cancer cells. The cytotoxic immune response is enhanced by co-administration of interferon, checkpoint inhibitor antibody and/or ADC.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/497,931, filed Apr. 26, 2017, which was a continuation of U.S. patentapplication Ser. No. 15/169,903 (now issued U.S. Pat. No. 9,670,286),filed Jun. 1, 2016, which was a continuation of U.S. patent applicationSer. No. 14/600,560 (now issued U.S. Pat. No. 9,382,329), which claimedthe benefit under 35 U.S.C. 119(e) of provisional U.S. PatentApplication Nos. 61/942,752, filed Feb. 21, 2014, and 62/049,826, filedSep. 12, 2014. U.S. Ser. No. 14/600,560 was a continuation-in-part ofU.S. patent application Ser. No. 14/106,737 (now issued U.S. Pat. No.9,682,143), filed Dec. 14, 2013, which was a continuation-in-part ofU.S. patent application Ser. No. 13/966,450 (now issued U.S. Pat. No.9,315,567), filed Aug. 14, 2013, which claimed the benefit under 35U.S.C. 119(e) of provisional U.S. Patent Applications 61/682,965, filedAug. 14, 2012; 61/733,268, filed Dec. 4, 2012, and 61/807,998, filedApr. 3, 2013. Each priority application is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 29, 2014, isnamed IBC140US1_SL and is 62,950 bytes in size.

FIELD

The present invention concerns compositions and methods of use ofbispecific antibodies targeting Trop-2 and CD3, that are capable ofinducing an immune response against Trop-2 expressing cells, such asTrop-2⁻ cancer cells. Preferably, the bispecific antibody isadministered in combination with one or more other therapeutic agents,such as an antibody-drug conjugate, an interferon such as such asinterferon-α, interferon-β or interferon-λ, or a checkpoint inhibitorantibody. More preferably, the bispecific antibody is ananti-Trop-2×anti-CD3 antibody that is administered in combination withinterferon-α. Most preferably, the anti-Trop-2 antibody is a hRS7antibody. The compositions and methods are of use to treat Trop-2⁺tumors, such as carcinomas of the esophagus, pancreas, lung, stomach,colon and rectum, urinary bladder, breast, ovary, uterus, kidney andprostate, more preferably pancreatic cancer or gastric cancer. Inpreferred embodiments, the bispecific antibody is made as aDOCK-AND-LOCK® complex, in which the components are attached togetherusing the binding interaction between dimerization and docking domain(DDD) moieties from human protein kinase A (PKA) regulatory subunits andanchor domain (AD) moieties from AKAPs (A-kinase anchor proteins).However, other methods of making bispecific antibody complexes are knownand may be used. The bispecific antibody redirects effector T cells,monocytes, NK cells or neutrophils to target diseased cells or tissuesand induces an immune response against the target.

BACKGROUND

Use of bispecific antibodies (bsAbs) to redirect effector T cells forthe targeted killing of tumor cells has shown considerable promise bothpre-clinically and clinically (see, e.g., Topp et al., 2012, Blood120:5185-87; Bargou et al., 2008, Science 321:974-77). The bispecificantibodies developed to date contain a first binding site specific toCD3 for T-cell recruitment and activation and a second binding site fora targeted disease-associated antigen, such as CD19 (Bassan, 2012, Blood120:5094-95). The bispecific antibody brings CD3⁺ T cells into directcontact with targeted disease cells and induces cell-mediatedcytotoxicity (Bassan, 2012). Anti-CD3×anti-CD19 bispecific antibodieshave been reported to produce a complete and durable molecular remissionat very low concentrations in approximately 70% of adult patients withMRD⁻ ALL (Topp et al., 2012, Blood 120:5185-87). Bispecific antibodiesrecognizing gliomas and the CD3 epitope on T cells have beensuccessfully used in treating brain tumors in human patients (Nitta, etal. Lancet 1990; 355:368-371).

Leukocyte redirecting bsAbs are not limited to T cells. The bispecifickiller engagers (BiKEs) comprising scFvs against the NK cell antigenCD16 and a tumor-associated antigen (e.g., CD19, CD22, CD33) have alsoshown potent anti-cancer activity (e.g., Miller, Hematology Soc HematolEduc Pogram 2013: 247-53). Other alternatives include trispecific killerengagers (TriKEs), such as anti-CD16×anti-CD19×anti-CD22 (Miller, 2013;Gleason et al., 2012, Mol Cancer Ther 11:2674-84). Ananti-CD16×anti-CD33 BiKE was used to treat AML and myelodysplasticsyndrome (Miller, 2013; Wiernik et al., 2013, Clin Cancer Res19:3844-55). In refractory AML, a CD16×CD33 BiKE led to potent tumorcell killing and cytokine production by NK cells. Inhibition of ADAM17enhanced the CD16×CD33 BiKE response (Miller, 2013). Other trispecific,trivalent constructs, for example against CD16/CD19/HLA-DR, have beenreported (Schubert et al., 2012, mAbs 4:45-56).

Numerous methods to produce bispecific antibodies are known (see, e.g.U.S. Pat. No. 7,405,320). Bispecific antibodies can be produced by thequadroma method, which involves the fusion of two different hybridomas,each producing a monoclonal antibody recognizing a different antigenicsite (Milstein and Cuello, Nature 1983; 305:537-540). The fusedhybridomas are capable of synthesizing two different heavy chains andtwo different light chains, which can associate randomly to give aheterogeneous population of 10 different antibody structures of whichonly one of them, amounting to ⅛ of the total antibody molecules, willbe bispecific, and therefore must be further purified from the otherforms. Fused hybridomas are often less stable cytogenetically than theparent hybridomas, making the generation of a production cell line moreproblematic.

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies, so that the resulting hybrid conjugate will bindto two different targets (Staerz, et al. Nature 1985; 314:628-631;Perez, et al. Nature 1985; 316:354-356). Bispecific antibodies generatedby this approach are essentially heteroconjugates of two IgG molecules,which diffuse slowly into tissues and are rapidly removed from thecirculation. Bispecific antibodies can also be produced by reduction ofeach of two parental monoclonal antibodies to the respective halfmolecules, which are then mixed and allowed to reoxidize to obtain thehybrid structure (Staerz and Bevan. Proc Natl Acad Sci USA 1986;83:1453-1457). An alternative approach involves chemically cross-linkingtwo or three separately purified Fab′ fragments using appropriatelinkers. All these chemical methods are undesirable for commercialdevelopment due to high manufacturing cost, laborious productionprocess, extensive purification steps, low yields (<20%), andheterogeneous products.

Discrete V_(H) and V_(L) domains of antibodies produced by recombinantDNA technology may pair with each other to form a dimer (recombinant Fvfragment) with binding capability (U.S. Pat. No. 4,642,334). However,such non-covalently associated molecules are not sufficiently stableunder physiological conditions to have any practical use. Cognate V_(H)and V_(L) domains can be joined with a peptide linker of appropriatecomposition and length (usually consisting of more than 12 amino acidresidues) to form a single-chain Fv (scFv) with binding activity.Methods of manufacturing scFv-based agents of multivalency andmultispecificity by varying the linker length were disclosed in U.S.Pat. Nos. 5,844,094, 5,837,242 and WO 98/44001. Common problems thathave been frequently associated with generating scFv-based agents ofmultivalency and multispecificity are low expression levels,heterogeneous products, instability in solution leading to aggregates,instability in serum, and impaired affinity.

Several bispecific antibodies targeting CD3 and CD19 are in clinicaldevelopment. An scFv-based bispecific antibody construct, known as BITE®(Bispecific T-cell Engager), employs a single polypeptide containing 2antigen-binding specificities, each contributed by a cognate VH and VL,linked in tandem via a flexible linker (see, e.g., Nagorsen et al.,2009, Leukemia & Lymphoma 50:886-91; Amann et al., 2009, J Immunother32:453-64; Baeuerle and Reinhardt, 2009, Cancer Res 69:4941-44). Anotherbispecific antibody called DART® (Dual-Affinity Re-Targeting) utilizes adisulfide-stabilized diabody design (see, e.g., Moore et al., 2011,Blood 117:4542-51; Veri et al., 2010, Arthritis Rheum 62:1933-43). BothBITE® and DART® exhibit fast blood clearance due to their small size(˜55 kDa), which requires frequent administration to maintaintherapeutic levels of the bispecific antibodies.

Interferons are critical role players in the antitumor and antimicrobialhost defense, and have been extensively explored as therapeutic agentsfor cancer and infectious disease (Billiau et al., 2006, Cytokine GrowthFactor Rev 17:381-409; Pestka et al., 2004, Immunol Rev 202:8-32).Despite considerable efforts with type I and II interferons (IFN-α/β andγ), their use in clinic settings have been limited because of the shortcirculation half-life, systemic toxicity, and suboptimal responses inpatients (Pestka et al., 2004, Immunol Rev 202:8-32; Miller et al.,2009, Ann N Y Acad Sci 1182:69-79). The discovery of the IFN-λ family inearly 2003 brought an exciting new opportunity to develop alternativeIFN agents for these unmet clinical indications (Kotenko et al., 2003,Nat Immunol 4:69-77; Sheppard et al., 2003, Nat Immunol 4:63-8).

The therapeutic effectiveness of IFNs has been validated to date by theapproval of IFN-α2 for treating hairy cell leukemia, chronic myelogenousleukemia, malignant melanoma, follicular lymphoma, condylomataacuminata, AIDs-related Kaposi sarcoma, and chronic hepatitis B and C;IFN-β for treating multiple sclerosis; and IFN-γ for treating chronicgranulomatous disease and malignant osteopetrosis. Despite a vastliterature on this group of autocrine and paracrine cytokines, theirfunctions in health and disease are still being elucidated, includingmore effective and novel forms being introduced clinically (Pestka,2007, J. Biol. Chem 282:20047-51; Vilcek, 2006, Immunity 25:343-48). Theeffects of combination of various interferons with antibody-basedtherapies also remain under investigation.

Antibody-drug conjugates (ADCs) are a potent class of therapeuticconstructs that allow targeted delivery of cytotoxic agents to targetcells, such as cancer cells. Because of the targeting function, thesecompounds show a much higher therapeutic index compared to the samesystemically delivered agents. ADCs have been developed as intactantibodies or antibody fragments, such as scFvs. The antibody orfragment is linked to one or more copies of drug via a linker that isstable under physiological conditions, but that may be cleaved onceinside the target cell. ADCs approved for therapeutic use includegemtuzumab ozogamicin for AML (subsequently withdrawn from the market),brentuximab vedotin for ALCL and Hodgkin lymphoma, and trastuzumabemtansine for HER2-positive metastatic breast cancer (Verma et al.,2012, N Engl J Med 367:1783-91; Bross et al., 2001, Clin Cancer Res7:1490-96; Francisco et al., 2003, Blood 102:1458-65). Numerous othercandidate ADCs are currently in clinical testing, such as inotuzumabozogamicin (Pfizer), glembatumomab vedotin (Celldex Therapeutics),SAR3419 (Sanofi-Aventis), SAR56658 (Sanofi-Aventis), AMG-172 (Amgen),AMG-595 (Amgen), BAY-94-9343 (Bayer), BIIB015 (Biogen Idec), BT062(Biotest), SGN-75 (Seattle Genetics), SGN-CD19A (Seattle Genetics),vorsetuzumab mafodotin (Seattle Genetics), ABT-414 (AbbVie), ASG-5ME(Agensys), ASG-22ME (Agensys), ASG-16M8F (Agensys), IMGN-529(ImmunoGen), IMGN-853 (ImmunoGen), MDX-1203 (Medarex), MLN-0264(Millenium), RG-7450 (Roche/Genentech), RG-7458 (Roche/Genentech),RG-7593 (Roche/Genentech), RG-7596 (Roche/Genentech), RG-7598(Roche/Genentech), RG-7599 (Roche/Genentech), RG-7600 (Roche/Genentech),RG-7636 (Roche/Genentech), anti-PSMA ADC (Progenics), lorvotuzumabmertansine (ImmunoGen), milatuzumab-doxorubicin (Immunomedics), IMMU-130(Immunomedics), IMMU-132 (Immunomedics) and antibody conjugates ofpro-2-pyrrolinodoxorubicin. (See, e.g., Li et al., 2013, Drug Disc Ther7:178-84; Firer & Gellerman, J Hematol Oncol 5:70; Beck et al., 2010,Discov Med 10:329-39; Mullard, 2013, Nature Rev Drug Discovery 12:329,Provisional U.S. Patent Application 61/761,845.) Because of thepotential of ADCs to act as potent anti-cancer agents with reducedsystemic toxicity, they may be used either alone or as an adjuncttherapy to reduce tumor burden.

Another promising approach to immunotherapy concerns use of antagonisticantibodies against immune checkpoint proteins (e.g., Pardoll, 2012,Nature Reviews Cancer 12:252-64). Immune checkpoints function asendogenous inhibitory pathways for immune system function that act tomaintain self-tolerance and to modulate the duration and extent ofimmune response to antigenic stimulation (Pardoll, 2012). However, itappears that tumor tissues and possibly certain pathogens may co-opt thecheckpoint system to reduce the effectiveness of host immune response,resulting in tumor growth and/or chronic infection (see, e.g., Pardoll,2012, Nature Reviews Cancer 12:252-64; Nirschl & Drake, 2013, ClinCancer Res 19:4917-24). Checkpoint molecules include CTLA4 (cytotoxic Tlymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1(programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3),TIM-3 (T cell immunoglobulin and mucin protein-3) and several others(Pardoll, 2012, Nature Reviews Cancer 12:252-64; Nirschl & Drake, 2013,Clin Cancer Res 19:4917-24). Antibodies against several of thecheckpoint proteins (CTLA4, PD1, PD-L1) are in clinical trials and hasshown unexpected efficacy againts tumors that were resistant to standardtreatments.

A need exists for methods and compositions to generate improvedbispecific antibody complexes with longer T_(1/2), betterpharmacokinetic properties, increased in vivo stability and/or improvedin vivo efficacy. A further need exists for combination therapies toimprove efficacy of treatments directed to inducing immune responseagainst various diseases, such as Trop-2⁺ cancer.

SUMMARY

The present invention relates to bispecific antibodies of use to treatdiseases involving Trop-2+ cells, such as Trop-2⁺ cancer cells. Trop-2is overexpressed in numerous types of solid tumors, such as carcinomasof the esophagus, pancreas, lung, stomach, colon and rectum, urinarybladder, breast, ovary, uterus, cervix, kidney and prostate. Preferably,the bispecific antibody is of use to treat gastric cancer or pancreaticcancer. Administration of the bispecific antibody induces an immuneresponse to cells that are Trop-2⁺. Although Trop-2 is also expressed insome normal tissues (e.g., Stepan et al., 2011, J Histochem Cytochem59:701-10), the Examples below demonstrate that anti-Trop-2 antibodiesmay be administered in vivo in both animal model systems and humansubjects, with only tolerable toxicities. In other preferredembodiments, administration of bispecific antibody to a subject inducesan immune response against Trop-2⁺ cancer cells without elevating levelsof cytokines that would induce cytokine release syndrome (CRS). Inalternative preferred embodiments, the bispecific antibody inducestrogocytosis of cell surface antigens between Trop-2⁺ cancer cells and Tcells.

In preferred embodiments, the bispecific antibody contains binding sitesfor Trop-2 and for CD3. However, other T cell or leukocyte antigens maybe targeted besides CD3. Exemplary T-cell antigens are selected from thegroup consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30,CD40, CD40L, CD44, CD45, CD69 and CD90. Exemplary antigens expressed onNK cells are selected from the group consisting of CD8, CD16, CD56,CD57, ADAM17, KIR and CD137. Exemplary monocyte antigens are selectedfrom the group consisting of CD74, HLA-DR alpha chain, CD14, CD16, CD64and CD89. Exemplary neutrophil antigens are selected from the groupconsisting of CEACAM6, CEACAM8, CD16b, CD32a, CD89, CD177, CD11a, CD11band SLC44A2. Preferably the T-cell antigen is CD3, or the NK cellantigen is CD16.

In alternative embodiments, other tumor-associated antigens besidesTrop-2 may be targeted. Tumor-associated antigens that may be targetedinclude, but are not limited to, alpha-fetoprotein (AFP), α-actinin-4,A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE,BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m,CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33,CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64,CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD126,CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA4, CXCR4,CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEA (CEACAM5),CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M,Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor,G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionicgonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia induciblefactor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ,IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12,IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1),KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migrationinhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3,mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13,MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, pancreaticcancer mucin, PD1 receptor, placental growth factor, p53, PLAGL2,prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6,IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC,TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras, an oncogene marker andan oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207).

Exemplary anti-TAA antibodies that may be used include, but are notlimited to, hA19 (anti-CD19, U.S. Pat. No. 7,109,304), hR1 (anti-IGF-1R,U.S. patent application Ser. No. 12/722,645, filed Mar. 12, 2010), hPAM4(anti-MUC5ac, U.S. Pat. No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No.7,251,164), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496),the Examples section of each cited patent or application incorporatedherein by reference.

Alternative antibodies that may be used for treatment of various diseasestates include, but are not limited to, abciximab (anti-glycoproteinIIb/IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab(anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20),panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20),trastuzumab (anti-ErbB2), lambrolizumab (anti-PD1 receptor), nivolumab(anti-PD1 receptor), ipilimumab (anti-CTLA4), abagovomab (anti-CA-125),adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab(anti-CD125), obinutuzumab (GA101, anti-CD20), CC49 (anti-TAG-72),AB-PG1-XG1-026 (anti-PSMA, U.S. patent application Ser. No. 11/983,372,deposited as ATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO2009/130575), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25),daclizumab (anti-CD25), efalizumab (anti-CD11a), GA101 (anti-CD20;Glycart Roche), atalizumab (anti-α4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), BENLYSTA® (HumanGenome Sciences); anti-CD38 antibodies such as MOR03087 (MorphoSys AG),MOR202 (Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson &Johnson).

Preferably, the bispecific antibody is administered in combination withone or more other therapeutic agents, such as antibodies, antibodyfragments, peptides, drugs, toxins, chemotherapeutic agents, enzymes,nucleases, hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes. More preferably, the additional therapeuticagent is an antibody-drug conjugate, an interferon such as such asinterferon-α, interferon-β or interferon-λ, or an antagonisticcheckpoint inhibitor antibody. Most preferably, the therapeutic agent isinterferon-α.

An exemplary design for a leukocyte redirecting bsAb disclosed in theExamples below combined an anti-CD3 scFv with an anti-CD19 F(ab)₂ toform a construct designated (19)-3s, which specifically targeted Bcells. Other bsAbs combining anti-CD3 with antibody fragments againstother tumor-associated antigens, discussed in more detail below, are ofuse in targeted leukocyte immunotherapy of various solid tumors. Theadvantages of this design include bivalent binding to tumor cells, alarger size (˜130 kDa) to preclude rapid renal clearance, and potentleukocyte mediated cytotoxicity. The bsAbs mediate the formation ofimmunological synapses between leukocytes and cognate target cells,induce leukocyte activation and proliferation in the presence of targetcells, redirect potent leukocyte mediated killing of target cells invitro and inhibit growth of human tumors in vivo.

A preferred embodiment concerns leukocyte redirecting bispecificantibodies produced as trivalent DNL® complexes, with longer T_(1/2),better pharmacokinetic properties and increased in vivo stability.Methods for production and use of DNL® complexes, comprising dimers ofDDD moieties from human PKA regulatory subunits RIα, RIβ, RIIα or RIIβ,bound to AD moieties from AKAPs, are well known (see, e.g., U.S. Pat.Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787; 7,666,400; 7,906,118;7,901,680; 8,003,111 and 8,034,352, the Examples section of eachincorporated herein by reference.) By attaching different effectormoieties, such as antibodies or antibody fragments, to the DDD and ADmoieties, DNL® complexes comprising virtually any combination ofeffectors may be constructed and used.

The antibodies of use can be of various isotypes, preferably human IgG1,IgG2, IgG3 or IgG4, more preferably comprising human IgG1 hinge andconstant region sequences. The antibodies or fragments thereof can bechimeric human-mouse, humanized (human framework and murinehypervariable (CDR) regions), or fully human, as well as variationsthereof, such as half-IgG4 antibodies (referred to as “unibodies”), asdescribed by van der Neut Kolfschoten et al. (Science 2007;317:1554-1557). More preferably, the antibodies or fragments thereof maybe designed or selected to comprise human constant region sequences thatbelong to specific allotypes, which may result in reduced immunogenicitywhen administered to a human subject. Preferred allotypes foradministration include a non-G1m1 allotype (nG1m1), such as G1m3,G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selectedfrom the group consisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

Other preferred embodiments concern compositions and/or use ofleukocyte-redirecting complexes in combination with one or morecheckpoint inhibitor antibodies. Such antibodies are antagonistic forcheckpoint inhibitor function. Many such antibodies are known in theart, such as lambrolizumab (MK-3475, Merck), nivolumab (BMS-936558,Bristol-Myers Squibb), pidilizumab (CT-011, CureTech Ltd.), AMP-224(Merck), MDX-1105 (Medarex), MEDI4736 (MedImmune), MPDL3280A(Genentech), BMS-936559 (Bristol-Myers Squibb), ipilimumab(Bristol-Myers Squibb) and tremelimumab (Pfizer). Anti-KIR antibodiessuch as lirlumab (Innate Pharma) and IPH2101 (Innate Pharma) may performsimilar functions in NK cells. Any known checkpoint inhibitor antibodymay be used in combination with one or more of the other agents.Combination therapy with immunostimulatory antibodies has been reportedto enhance efficacy, for example against tumor cells. Morales-Kastresanaet al. (2013, Clin Cancer Res 19:6151-62) showed that the combination ofanti-PD-L1 (10B5) antibody with anti-CD137 (1D8) and anti-OX40 (OX86)antibodies provided enhanced efficacy in a transgenic mouse model ofhepatocellular carcinoma. Combination of anti-CTLA4 and anti-PD1antibodies has also been reported to be highly efficacious (Wolchok etal., 2013, N Engl J Med 369:122-33). Combination of rituximab withanti-KIR antibody, such as lirlumab (Innate Pharma) or IPH2101 (InnatePharma), was also more efficacious against hematopoietic tumors (Kohrtet al., 2012). The person of ordinary skill will realize that thesubject combination therapy may include combinations with multipleantibodies that are immunostimulatory, anti-tumor or anti-infectiousagent.

Another agent that may be used in combination is an interferon.Interferons of use are known in the art and may include interferon-α,interferon-β, interferon-λ1, interferon-λ2 or interferon-λ3. Preferably,the interferon is interferon-α. The subject interferon may beadministered as free interferon, PEGylated interferon, an interferonfusion protein or interferon conjugated to an antibody.

In alternative embodiments, one or more of the immunomodulatory agentsdiscussed above may be used in combination with an antibody-drugconjugate (ADC). ADCs are particularly effective for reducing tumorburden without significant systemic toxicity and may act to improve theeffectiveness of the immune response induced by leukocyte retargetingbsAb, interferon and/or checkpoint inhibitor antibody. Exemplary ADCs ofuse may include ADCs approved for therapeutic use include gemtuzumabozogamicin for AML (subsequently withdrawn from the market), brentuximabvedotin for ALCL and Hodgkin lymphoma, and trastuzumab emtansine forHER2-positive metastatic breast cancer (Verma et al., 2012, N Engl J Med367:1783-91; Bross et al., 2001, Clin Cancer Res 7:1490-96; Francisco etal., 2003, Blood 102:1458-65). Numerous other candidate ADCs arecurrently in clinical testing, such as inotuzumab ozogamicin (Pfizer),glembatumomab vedotin (Celldex Therapeutics), SAR3419 (Sanofi-Aventis),SAR56658 (Sanofi-Aventis), AMG-172 (Amgen), AMG-595 (Amgen), BAY-94-9343(Bayer), BIIB015 (Biogen Idec), BT062 (Biotest), SGN-75 (SeattleGenetics), SGN-CD19A (Seattle Genetics), vorsetuzumab mafodotin (SeattleGenetics), ABT-414 (AbbVie), ASG-5ME (Agensys), ASG-22ME (Agensys),ASG-16M8F (Agensys), IMGN-529 (ImmunoGen), IMGN-853 (ImmunoGen),MDX-1203 (Medarex), MLN-0264 (Millenium), RG-7450 (Roche/Genentech),RG-7458 (Roche/Genentech), RG-7593 (Roche/Genentech), RG-7596(Roche/Genentech), RG-7598 (Roche/Genentech), RG-7599 (Roche/Genentech),RG-7600 (Roche/Genentech), RG-7636 (Roche/Genentech), anti-PSMA ADC(Progenics), lorvotuzumab mertansine (ImmunoGen),milatuzumab-doxorubicin (Immunomedics), IMMU-130 (Immunomedics) andIMMU-132 (Immunomedics). (See, e.g., Li et al., 2013, Drug Disc Ther7:178-84; Firer & Gellerman, J Hematol Oncol 5:70; Beck et al., 2010,Discov Med 10:329-39; Mullard, 2013, Nature Rev Drug Discovery 12:329.)Preferably, where an ADC is used in combination with an immunomodulator,the ADC is administered prior to the immunomodulator.

The subject agents may be administered in combination with one or moreother immunomodulators to enhance the immune response. Immunomodulatorsmay include, but are not limited to, a cytokine, a chemokine, a stemcell growth factor, a lymphotoxin, an hematopoietic factor, a colonystimulating factor (CSF), erythropoietin, thrombopoietin, tumor necrosisfactor-α (TNF), TNF-β, granulocyte-colony stimulating factor (G-CSF),granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, interferon-λ, stem cell growth factordesignated “S1 factor”, human growth hormone, N-methionyl human growthhormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin,proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepaticgrowth factor, prostaglandin, fibroblast growth factor, prolactin,placental lactogen, OB protein, mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, NGF-β, platelet-growth factor, TGF-α, TGF-β,insulin-like growth factor-I, insulin-like growth factor-II,macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin,endostatin, or lymphotoxin. In certain embodiments, aleukocyte-redirecting bispecific antibody or antibody fragment may beattached to an immunomodulator, such as a cytokine. Cytokine complexesare disclosed, for example, in U.S. Pat. Nos. 7,906,118 and 8,034,3522,the Examples section of each incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1. Schematic diagram of formation of DOCK-AND-LOCK® complexcomprising anti-CD19 F(ab)₂×anti-CD3 scFv.

FIG. 2A. Immune synapse formation between Daudi Burkitt lymphoma and Tcells, mediated by (19)-3s. Freshly isolated T cells were combined withDaudi cells at an E:T ratio of 2.5:1. Cells were treated with 0, 1 or 5μg/mL of (19)-3s for 30 min at room temperature prior to analysis byflow cytometry. Anti-CD20-FITC and anti-CD7-APC were used to identifyDaudi and T cells, respectively. Co-binding was indicated as the % ofCD20⁺/CD7⁺ events. After treatment with (19)-3s, 45.5% of flow eventswere CD20/CD7 dual-positive, indicating synapsed Daudi and T cells.

FIG. 2B. Conditions were as in FIG. 2(A), except for the absence of(19)-3s antibody. Compared to FIG. 2(A), only 2% of flow events wereCD20/CD7 dual-positive without antibody.

FIG. 2C. Addition of (19)-3s resulted in association of >90% of theDaudi with T cells.

FIG. 3A. Jurkat (T cells) and Daudi (B cells) were combined at a 1:1ratio, treated with 0.1 μg/mL (19)-3s for 30 minutes and stained withanti-CD20-FITC, prior to analysis by fluorescence microscopy.

FIG. 3B. Jurkat (T cells) and Daudi (B cells) were combined at a 1:1ratio, treated with 0.1 μg/mL (19)-3s for 30 minutes and stained withanti-CD20-FITC and anti-CD3-PE, prior to analysis by fluorescencemicroscopy.

FIG. 3C. The merged image of FIGS. 3A and 3B reveals synapse formationbetween green-stained Daudi and red-stained Jurkat cells.

FIG. 3D. Synapse formation was not evident in the absence of (19)-3s.

FIG. 4. Dose response analysis of (19)-3s mediated cell-to-cellassociation of Daudi and Jurkat cells as a function of increasingconcentrations of (19)-3s.

FIG. 5A. Comparison of cell-to-cell association mediated by BITE® andDART™. The data for BITE® and DART™ was taken from Moore et al. (2011,Blood 117:4542-4551.

FIG. 5B. Comparison of cell-to-cell association mediated by (19)-3s.

FIG. 6A. Synapse formation between T cells and Capan-1 pancreatic cancercells mediated by (19)-3s control bsAb. CFSE-labeled Capan-1 cells werecoincubated with PKH26-labeled Jurkat in the presence of the bsAb.

FIG. 6B. Synapse formation between T cells and Capan-1 pancreatic cancercells mediated by (M1)-3s MUC5AC bsAb. CFSE-labeled Capan-1 cells werecoincubated with PKH26-labeled Jurkat in the presence of the bsAb.

FIG. 6C. Synapse formation between T cells and Capan-1 pancreatic cancercells mediated by (E1)-3s TROP-2 targeting bsAb. CFSE-labeled Capan-1cells were coincubated with PKH26-labeled Jurkat in the presence of thebsAb.

FIG. 7A. T-cell activation by (19)-3s. Upregulation of CD69 expressionis an early event in T-cell activation. Daudi cells combined with PBMCswere treated overnight with the indicated antibodies, and stained withanti-CD3-PE and anti-CD69-APC, prior to analysis by flow cytometry. CD69expression was evaluated following gating of T cells by forward vs. sidescattering and anti-CD3 staining. Combination of Daudi cells with anequal number of PBMCs resulted in 1.6% CD69+ T cells. Addition of 3ng/mL (19)-3s induced 27% CD69+ T cells. Neither a control construct[(M1)-3s], which comprises the Okt3-scFv-AD2 module fused with anon-targeting F(ab)₂, nor the hA19-Fab-DDD2 module, induced T-cellactivation.

FIG. 7B. T-cell activation by (19)-3s. Daudi cells combined withpurified T cells were treated overnight with the indicated antibodies,and stained with anti-CD3-PE and anti-CD69-APC, prior to analysis byflow cytometry. CD69 expression was evaluated following gating of Tcells by forward vs. side scattering and anti-CD3 staining. Treatment ofDaudi and purified T cells with (M1)-3s or hA19-Fab-DDD2 did notincrease the number of CD69+ T cells (<4%), compared to the untreatedcell mixture. Alternatively, (19)-3s induced robust T-cell activation,producing 80% CD69+ cells.

FIG. 7C. T-cell activation by (19)-3s. Purified T cells alone weretreated overnight with the indicated antibodies, and stained withanti-CD3-PE and anti-CD69-APC, prior to analysis by flow cytometry. CD69expression was evaluated following gating of T cells by forward vs. sidescattering and anti-CD3 staining. Without the addition of Daudi (target)cells, (19)-3s did not induce CD69 expression and T-cell activation.These results demonstrate that (19)-3s-mediated synapse formationbetween T cells and target cells is both required and sufficient forT-cell activation.

FIG. 8A. Induction of T-cell proliferation by (19)-3s. PBMCs wereincubated with 3 nM or 30 pM of (19)-3s, compared to IL-2/PHA positivecontrol and (14)-3s (non-target-binding control).

FIG. 8B. Induction of T-cell proliferation by (19)-3s. T cellproliferation was not observed in PBMCs depleted of B cells, indicatingthat target cells (B cells) are required for T-cell activation andproliferation.

FIG. 9A. In vitro cytotoxicity of (19)-3s T-cell redirecting bsAbs.Dose-response curves for cytotoxicity to Nalm-6, Raji, Ramos and Namalwacancer cells were determined for the (19)-3s DNL® bsAb complex.

FIG. 9B. In vitro cytotoxicity of (19)-3s T-cell redirecting bsAbs.Dose-response curves for cytotoxicity to Nalm-6, Raji, Ramos and Namalwacancer cells were determined for the (14)-3s (non-targeting) DNL® bsAbcomplex.

FIG. 9C. Consistent results were observed using PBMCs, or T cells,obtained from two different donors and Nalm-6 cancer cells.

FIG. 10A. In vitro cytotoxicity of (20)-3s, (22)-3s and (C2)-3s T-cellredirecting bsAbs. Dose-response curves were determined for cytotoxicityto Namalwa cells induced by (20)-3s, (22)-3s and (C2)-3s T-cellredirecting bsAbs.

FIG. 10B. In vitro cytotoxicity of (20)-3s, (22)-3s and (C2)-3s T-cellredirecting bsAbs. Dose-response curves were determined for cytotoxicityto Jeko cells induced by (20)-3s, (22)-3s and (C2)-3s T-cell redirectingbsAbs.

FIG. 10C. In vitro cytotoxicity of (20)-3s, (22)-3s and (C2)-3s T-cellredirecting bsAbs. Dose-response curves were determined for cytotoxicityto Daudi cells induced by (20)-3s, (22)-3s and (C2)-3s T-cellredirecting bsAbs.

FIG. 11A. In vitro cytotoxicity of T-cell redirecting bsAbs in solidtumor cell lines. Dose-response curves were determined for cytotoxicityto the LS174T colon adenocarcinoma cell line for the (14)-3s bsAb,compared to non-targeting (19)-3s bsAb.

FIG. 11B. In vitro cytotoxicity of T-cell redirecting bsAbs in solidtumor cell lines. Dose-response curves were determined for cytotoxicityto the Capan-1 pancreatic adenocarcinoma cell line for the (E1)-3s bsAb,compared to non-targeting (19)-3s bsAb.

FIG. 11C. In vitro cytotoxicity of T-cell redirecting bsAbs in solidtumor cell lines. Dose-response curves were determined for cytotoxicityto the NCI-N87 gastric carcinoma cell line for the (E1)-3s and (15)-3sbsAbs, compared to non-targeting (19)-3s bsAb.

FIG. 12. Summary of in vitro cytotoxicity data for T-cell redirectingbsAbs in cancer cell lines.

FIG. 13A. In vivo retargeting of Raji lymphoma xenografts using (19)-3sbsAb. NOD/SCID mice bearing Raji Burkitt lymphoma (1×10⁶ cells)xenografts, reconstituted with human PBMCs (5×10⁶ cells) and treatedwith (19)-3s for only 1 week, administered as indicated by the arrows.Control with untreated cells.

FIG. 13B. In vivo retargeting of Raji lymphoma xenografts using (19)-3sbsAb. NOD/SCID mice bearing Raji Burkitt lymphoma (1×10⁶ cells)xenografts, reconstituted with human PBMCs (5×10⁶ cells) and treatedwith (19)-3s for only 1 week, administered as indicated by the arrows.Cells were treated with a single dose of 130 μg.

FIG. 13C. In vivo retargeting of Raji lymphoma xenografts using (19)-3sbsAb. NOD/SCID mice bearing Raji Burkitt lymphoma (1×10⁶ cells)xenografts, reconstituted with human PBMCs (5×10⁶ cells) and treatedwith (19)-3s for only 1 week, administered as indicated by the arrows.Cells were treated 3× with 43 μg per dose.

FIG. 13D. In vivo retargeting of Raji lymphoma xenografts using (19)-3sbsAb. NOD/SCID mice bearing Raji Burkitt lymphoma (1×10⁶ cells)xenografts, reconstituted with human PBMCs (5×10⁶ cells) and treatedwith (19)-3s for only 1 week, administered as indicated by the arrows.Cells were treated 5× with 26 μg per dose.

FIG. 14A. Effect of repeated dosing on in vivo retargeting of Rajilymphoma xenografts using (19)-3s bsAb. NOD/SCID mouse xenografts wereprepared as indicated in the legend to FIG. 13. The (19)-3s wasadministered as indicated by the arrows. FIG. 14A shows untreatedcontrol.

FIG. 14B. Effect of repeated dosing on in vivo retargeting of Rajilymphoma xenografts using (19)-3s bsAb. The (19)-3s was administered asindicated by the arrows. Cells were treated 2× with 130 μg per dose of(19)-3s administered i.v.

FIG. 14C. Effect of repeated dosing on in vivo retargeting of Rajilymphoma xenografts using (19)-3s bsAb. The (19)-3s was administered asindicated by the arrows. Cells were treated treated 2× with 130 μg perdose of (19)-3s administered s.c.

FIG. 14D. Effect of repeated dosing on in vivo retargeting of Rajilymphoma xenografts using (19)-3s bsAb. The (19)-3s was administered asindicated by the arrows. Cells were treated treated 4× with 65 μg perdose of (19)-3s administered i.v.

FIG. 14E. Effect of repeated dosing on in vivo retargeting of Rajilymphoma xenografts using (19)-3s bsAb. The (19)-3s was administered asindicated by the arrows. Cells were treated treated 6× with 43 μg perdose of (19)-3s administered i.v.

FIG. 14F. Effect of repeated dosing on in vivo retargeting of Rajilymphoma xenografts using (19)-3s bsAb. The (19)-3s was administered asindicated by the arrows. Cells were treated treated 6× with 43 μg perdose of control (M1)-3s administered i.v.

FIG. 15A. In vivo efficacy of T-cell retargeting bsAbs in solid tumorxenografts. NOD/SCID mouse xenografts were prepared with LS174T colonadenocarcinoma. Mice were administered T cells only without bsAb.

FIG. 15B. In vivo efficacy of T-cell retargeting bsAbs in solid tumorxenografts. NOD/SCID mouse xenografts were prepared with LS174T colonadenocarcinoma. Mice were treated with (E1)-3s bsAb as indicated.

FIG. 15C. In vivo efficacy of T-cell retargeting bsAbs in solid tumorxenografts. NOD/SCID mouse xenografts were prepared with Capan-1pancreatic carcinoma. Mice were administered PBMCs only without bsAb.

FIG. 15D. In vivo efficacy of T-cell retargeting bsAbs in solid tumorxenografts. NOD/SCID mouse xenografts were prepared with Capan-1pancreatic carcinoma. Mice were treated with (14)-3s bsAb as indicated.

FIG. 16A. In vivo inhibition of tumor growth by (E1)-3s DNL® complex inthe presence or absence of interferon-α. Capan-1 pancreatic carcinomaxenografts in NOD/SCID mice were treated with anti-TROP-2×anti-CD3 bsAbwith or without added interferon-α. The interferon-α was added in theform of a TROP-2 targeting DNL® complex.

FIG. 16B. In vivo inhibition of tumor growth by (E1)-3s DNL® complex inthe presence or absence of interferon-α. Capan-1 pancreatic carcinomaxenografts in NOD/SCID mice were treated with anti-TROP-2×anti-CD3 bsAbwith or without added interferon-α. The interferon-α was added as thecommercially available PEGASYS® (peginterferon alfa-2a).

FIG. 17. Survival curves for NOD/SCID mice treated with (E1)-3s with orwithout interferon-α. Controls were untreated or treated withinterferon-α alone.

FIG. 18. In vivo inhibition of tumor growth by (E1)-3s DNL® complex inthe presence or absence of interferon-α, compared to TF12 control.Capan-1 pancreatic carcinoma xenografts in NOD/SCID mice were treatedwith anti-TROP-2×anti-CD3 bsAb with or without added interferon-α, addedas PEGASYS®, compared to untreated control, TF12 control or PEGASYS®alone.

FIG. 19. Survival curves for NOD/SCID mice treated with (E1)-3s with orwithout interferon-α (PEGASYS®). Controls were untreated or treated withPEGASYS® alone or TF12 alone.

FIG. 20. In vivo inhibition of tumor growth by (E1)-3s DNL® complex inthe presence or absence of interferon-α, compared to TF12 control.NCI-N87 human gastric cancer xenografts in NOD/SCID mice were treatedwith anti-TROP-2×anti-CD3 bsAb with or without added interferon-α, addedas PEGASYS®, compared to untreated control, TF12 control or PEGASYS®alone.

FIG. 21. Survival curves for NOD/SCID mice with NCI-N87 gastric cancerxenografts treated with (E1)-3s with or without interferon-α (PEGASYS®).Controls were untreated or treated with PEGASYS® alone or TF12 alone.

FIG. 22. Schematic representation of the nascent E1-3 polypeptide. LP,leader peptide that is removed in mature protein; VH, heavy chainvariable domain, VK, kappa light chain variable domain, L1, linkerpeptide 1; L2, linker peptide 2; L3, linker pepide 3; 6H,hexa-histidine.

FIG. 23A. Ex vivo T cell redirected killing of BxPC3 human pancreaticcancer solid tumor cell line.

FIG. 23B. Ex vivo T cell redirected killing of Capan-1 human pancreaticcancer solid tumor cell line.

FIG. 23C. Ex vivo T cell redirected killing of NCI-N87 human gastriccancer solid tumor cell line.

FIG. 24. In vivo T cell redirected therapy of NCI-N87 gastric carcinomain NOD-SCID mice.

FIG. 25. Immunologic synapse formation and bidirectional trogocytosismediated by (E1)-3s. Purified T cells were mixed with BxPC3 cells at a5:1 ratio and incubated for 60 minutes with 0.1 nmol/L of the indicatedbsAb before staining with anti-Trop-2 MAb C518 and GAM-Fc-FITC. Thecells were analyzed by flow cytometry, with nonconjugated T cells andBxPC3 cells first gated by forward versus side scattering. Trogocytosisof Trop-2 from BxPC3 cells to T cells was evident by detection of Trop-2on T cells, specifically in cell mixtures with (E1)-3s, shown as thepercentage of Trop-2-positive unconjugated T cells.

FIG. 26. Immunologic synapse formation and bidirectional trogocytosismediated by (E1)-3s. Purified T cells were mixed with BxPC3 cells at a5:1 ratio and incubated for 60 minutes with 0.1 nmol/L of the indicatedbsAb before staining with anti-Trop-2 MAb C518 and GAM-Fc-FITC. Thecells were analyzed by flow cytometry, with nonconjugated T cells andBxPC3 cells first gated by forward versus side scattering. Trogocytosisresulted in a reduction of Trop-2 on BxPC3 cells, shown as the geometricMFI.

FIG. 27A. Cytokine induction. (A) PBMCs (6×10⁶ cells/well) were combinedwith Raji (5×10⁵ cells/well) and treated for 20 h with 0.1 nM 19-3 BiTE(checkered), (19)-3s (black), or incubated without bsAb (white, nottested for D-5). Concentrations of TNF-α, IFN-γ, IL-2, IL-6, and IL-10in the supernatant fluids were determined using commercial ELISA kits.D-1 through D-8 are independent blood donors, where only D-5 was used inboth A and B at the same time.

FIG. 27B. NCI-N87 cells (5×10⁵ cells/0.5 mL/well) were culturedovernight in 24-well plates to allow cell attachment. PBMCs were addedto wells containing attached NCI-N87 cells (10:1 ratio) and treated for20 h with 0.1 nM of (E1)-3s (black), peginterferonalfa-2a (white),(E1)-3s plus peginterferonalfa-2a (checkered), or untreated (gray).Concentrations of TNF-α, IFN-γ, IL-2, IL-6, and IL-10 in the supernatantfluids were determined using commercial ELISA kits. D-1 through D-8 areindependent blood donors, where only D-5 was used in both A and B at thesame time.

FIG. 28A. In-vitro cytotoxicity. Purified CD8⁺ T cells isolated from afirst donor were pre-treated for 24 h with 0.1 nM peginterferonalfa-2a(▴, dashed), 0.1 nM 20*-2b (●, grey) or media (▪, black) beforecombining with PKH-67 green fluorescent labeled NCI-N87 cells at a 5:1ratio. The cell mixtures were treated with titrations of (E1)-3s for twodays before counting the number of live NCI-N87 cells by flow cytometry.Non-linear regression analysis (sigmoidal dose-response) of the percentlysis, which was calculated for each sample using the formula:[1-(A₁/A₂)]×100, where A₁ and A₂ represent the number of viable targetcells in the test and untreated samples, respectively, vs the log of themolar concentration of (E1)-3s.

FIG. 28B. In-vitro cytotoxicity. Purified CD8⁺ T cells isolated from asecond donor were pre-treated for 24 h with 0.1 nM peginterferonalfa-2a(▴, dashed), 0.1 nM 20*-2b (●, grey) or media (▪, black) beforecombining with PKH-67 green fluorescent labeled NCI-N87 cells at a 5:1ratio. The cell mixtures were treated with titrations of (E1)-3s for twodays before counting the number of live NCI-N87 cells by flow cytometry.Non-linear regression analysis (sigmoidal dose-response) of the percentlysis, which was calculated for each sample using the formula:[1-(A₁/A₂)]×100, where A₁ and A₂ represent the number of viable targetcells in the test and untreated samples, respectively, vs the log of themolar concentration of (E1)-3s.

FIG. 29A. T-cell activation. Purified T cells were mixed 5:1 withNCI-N87 cells and treated for 18 h with (E1)-3s before measuring CD69expression by flow cytometry. Non-linear regression analysis (sigmoidaldose-response) of the percent CD69-positive CD4⁺ (●) or CD8⁺ (▪) T cellsvs the log of the molar concentration of (E1)-3s, in the presence(dashed line) or absence (solid line) of 0.1 nM peginterferonalfa-2a.

FIG. 29B. T-cell activation. Purified T cells were mixed 5:1 withNCI-N87 cells and treated for 18 h with (E1)-3s before measuring CD69expression by flow cytometry. Histogram showing anti-CD69-APC stainingof CD8⁺ T cells following treatment with 0.1 nM (E1)-3s (dotted), 0.1 nMpeginterferonalfa-2a (gray), or a combination of both agents (black), inthe presence of NCI-N87 cells.

FIG. 29C. T-cell activation. Purified T cells were mixed 5:1 withNCI-N87 cells and treated for 18 h with (E1)-3s before measuring CD69expression by flow cytometry. Percent CD69-positive CD8⁺ T cells afterincubation with 0.1 nM (E1)-3s (E) and/or 0.1 nM peginterferonalfa-2a(P), in the absence or presence of NCI-N87 target cells (T). Eachtreatment was assayed in triplicate. Error bars, S.D. *, P<0.001.

FIG. 29D. T-cell activation. Purified T cells were mixed 5:1 withNCI-N87 cells and treated for 18 h with (E1)-3s before measuring CD69expression by flow cytometry. Geometric mean fluorescence of the CD69⁺cells after incubation with 0.1 nM (E1)-3s (E) and/or 0.1 nMpeginterferonalfa-2a (P), in the absence or presence of NCI-N87 targetcells (T). Each treatment was assayed in triplicate. Error bars, S.D. *,P<0.001.

FIG. 30A. In-vivo efficacy with human pancreatic and gastric cancerxenografts. Groups of 8 mice inoculated with human T cells and Capan-1pancreatic cancer cells were treated daily for five days with 50 μg of(E1)-3s (▴, solid black) or 60 μg TF12 (▾, gray), once weekly for fourweeks with 0.6 μg of peginterferonalfa-2a (

, solid black), a combination of (E1)-3s and peginterferonalfa-2aregimens (●, solid black) or with saline (●, dashed black). Anadditional group was inoculated with Capan-1, but not T cells, andtreated with peginterferonalfa-2a (□, dashed black). Top panel,Kaplan-Meyer survival plots. Bottom panel, mean tumor volumes (+S.D.) vsdays. Data marked with an asterisk were adapted from FIG. 6C in Rossi etal. (2014, MAbs 6:381-91).

FIG. 30B. In-vivo efficacy with human pancreatic and gastric cancerxenografts. Groups of 8 mice inoculated with human T cells and Capan-1pancreatic cancer cells were treated daily for five days with 50 μg of(E1)-3s (▴, solid black) or 60 μg TF12 (▾, gray), once weekly for fourweeks with 0.6 μg of peginterferonalfa-2a (

, solid black), a combination of (E1)-3s and peginterferonalfa-2aregimens (●, solid black) or with saline (●, dashed black). Anadditional group was inoculated with Capan-1, but not T cells, andtreated with peginterferonalfa-2a (□, dashed black). Top panel,Kaplan-Meyer survival plots. Bottom panel, mean tumor volumes (+S.D.) vsdays. Data marked with an asterisk were adapted from FIG. 6C in Rossi etal. (2014, MAbs 6:381-91).

FIG. 30C. In-vivo efficacy with human pancreatic and gastric cancerxenografts. Groups of 8 mice inoculated with NCI-N87 gastric cancercells were treated daily for five days with 50 μg of (E1)-3s (▴, solidblack) or 60 μg TF12 (▾, gray), once weekly for four weeks with 0.6 μgof peginterferonalfa-2a (

, solid black), a combination of (E1)-3s and peginterferonalfa-2aregimens (●, solid black) or with saline (●, dashed black). Anadditional group was inoculated with Capan-1, but not T cells, andtreated with peginterferonalfa-2a (□, dashed black). Top panel,Kaplan-Meyer survival plots. Bottom panel, mean tumor volumes (+S.D.) vsdays. Data marked with an asterisk were adapted from FIG. 6C in Rossi etal. (2014, MAbs 6:381-91).

FIG. 31. Cytokine production induced by E1-3. PBMCs were combined at a5:1 ratio with BxPC-3 cells and treated with a titration of E1-3 for 24h. Cytokine concentrations were measured using Single-Analyte ELISArraykits (Qiagen). All cytokine levels were <10 pg/mL in the absence ofE1-3.

FIG. 32. In vitro redirected T cell killing of pancreatic and gastriccancer cell lines. Purified CD8⁺ T cells (1.2×10⁵/well) were mixed 6:1with target cells (2×10⁴/well) and treated with titrations of E1-3 in a96-well plate. After 48 h, wells were washed to remove T cells and theviable target cell densities were determined with an MTS assay. Exampleof results for one of several T cell donors.

FIG. 33A. In vivo therapy of human gastric tumor xenografts. PBMCs weremixed 2:1 with NCI-N87 cells and injected s.c. with matrigel in NOD-SCIDmice. Animals were given 50 μg E1-3 i.v. on Days 0 and 3. Mice weremonitored daily for signs of tumor out-growth, after which tumors weremeasured twice weekly with an endpoint measurement of >1.0 cm³. After176 days, 7 of 8 mice in the E1-3 treatment group had not reached theendpoint with 6 animals remaining tumor free.

FIG. 33B. In vivo therapy of human gastric tumor xenografts. PBMCs weremixed 2:1 with NCI-N87 cells and injected s.c. with matrigel in NOD-SCIDmice. Animals were given 50 μg E1-3 i.v. on Days 0 and 3. Mice weremonitored daily for signs of tumor out-growth, after which tumors weremeasured twice weekly with an endpoint measurement of >1.0 cm³. Tumorsin the control group comprising only PBMCs and NCI-87 reached the endpoint with a median time of 39.5 days.

DETAILED DESCRIPTION

Definitions

Unless otherwise specified, “a” or “an” means “one or more”.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, peptides, drugs, toxins, enzymes,nucleases, hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes.

An “antibody” as used herein refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. An“antibody” includes monoclonal, polyclonal, bispecific, multispecific,murine, chimeric, humanized and human antibodies.

A “naked antibody” is an antibody or antigen binding fragment thereofthat is not attached to a therapeutic or diagnostic agent. The Fcportion of an intact naked antibody can provide effector functions, suchas complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev50:59-87, 1998). Other mechanisms by which naked antibodies induce celldeath may include apoptosis. (Vaswani and Hamilton, Ann Allergy AsthmaImmunol 81: 105-119, 1998.)

An “antibody fragment” is a portion of an intact antibody such asF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFv, dAb and the like. Regardless ofstructure, an antibody fragment binds with the same antigen that isrecognized by the full-length antibody. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains or recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). “Single-chain antibodies”, often abbreviated as“scFv” consist of a polypeptide chain that comprises both a V_(H) and aV_(L) domain which interact to form an antigen-binding site. The V_(H)and V_(L) domains are usually linked by a peptide of 1 to 25 amino acidresidues. Antibody fragments also include diabodies, triabodies andsingle domain antibodies (dAb).

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains, including human framework region(FR) sequences. The constant domains of the antibody molecule arederived from those of a human antibody. To maintain binding activity, alimited number of FR amino acid residues from the parent (e.g., murine)antibody may be substituted for the corresponding human FR residues.

A “human antibody” is an antibody obtained from transgenic mice thathave been genetically engineered to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A human antibodyalso can be constructed by genetic or chromosomal transfection methods,as well as phage display technology, all of which are known in the art.(See, e.g., McCafferty et al., 1990, Nature 348:552-553 for theproduction of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see, e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.(See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which an antibody or antibodyfragment is linked to another protein or peptide, such as the same ordifferent antibody or antibody fragment or a DDD or AD peptide. Thefusion protein may comprise a single antibody component, a multivalentor multispecific combination of different antibody components ormultiple copies of the same antibody component. The fusion protein mayadditionally comprise an antibody or an antibody fragment and atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators and toxins. One preferred toxincomprises a ribonuclease (RNase), preferably a recombinant RNase. Apreferred immunomodulator might be an interferon, such as interferon-α,interferon-β or interferon-λ.

A “multispecific antibody” is an antibody that can bind simultaneouslyto at least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. A “multivalent antibody” is anantibody that can bind simultaneously to at least two targets that areof the same or different structure. Valency indicates how many bindingarms or sites the antibody has to a single antigen or epitope; i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyis able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Multispecific, multivalent antibodiesare constructs that have more than one binding site of differentspecificity.

A “bispecific antibody” is an antibody that can bind simultaneously totwo targets which are of different structure. Bispecific antibodies(bsAb) and bispecific antibody fragments (bsFab) may have at least onearm that specifically binds to, for example, a T cell, an NK cell, amonocyte or a neutrophil, and at least one other arm that specificallybinds to an antigen produced by or associated with a diseased cell,tissue, organ or pathogen, for example a tumor-associated antigen. Avariety of bispecific antibodies can be produced using molecularengineering.

An antibody preparation, or a composition described herein, is said tobe administered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient subject. In particular embodiments, anantibody preparation is physiologically significant if its presenceinvokes an antitumor response or mitigates the signs and symptoms of aninfectious disease state. A physiologically significant effect couldalso be the evocation of a humoral and/or cellular immune response inthe recipient subject leading to growth inhibition or death of targetcells.

Anti-Trop-2 Antibodies

In preferred embodiments, the subject bispecific antibody includes atleast one antibody or fragment thereof that binds to Trop-2. In a morepreferred embodiment, the anti-Trop-2 antibody may is a humanized RS7antibody (see, e.g., U.S. Pat. No. 7,238,785, incorporated herein byreference in its entirety), comprising the light chain CDR sequencesCDR1 (KASQDVSIAVA, SEQ ID NO:115); CDR2 (SASYRYT, SEQ ID NO:116); andCDR3 (QQHYITPLT, SEQ ID NO:117) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:118); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:119) and CDR3(GGFGSSYWYFDV, SEQ ID NO:120).

The RS7 antibody was a murine IgG₁ raised against a crude membranepreparation of a human primary squamous cell lung carcinoma. (Stein etal., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. (Stein et al., Int. J. CancerSupp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelialglycoprotein-1), but is also referred to as Trop-2.

Trop-2 is a type-I transmembrane protein and has been cloned from bothhuman (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells(Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to its roleas a tumor-associated calcium signal transducer (Ripani et al., Int JCancer 1998; 76:671-6), the expression of human Trop-2 was shown to benecessary for tumorigenesis and invasiveness of colon cancer cells,which could be effectively reduced with a polyclonal antibody againstthe extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5).

The growing interest in Trop-2 as a therapeutic target for solid cancers(Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is attested byfurther reports that documented the clinical significance ofoverexpressed Trop-2 in breast (Huang et al., Clin Cancer Res 2005;11:4357-64), colorectal (Ohmachi et al., Clin Cancer Res 2006;12:3057-63; Fang et al., Int J Colorectal Dis 2009; 24:875-84), and oralsquamous cell (Fong et al., Modern Pathol 2008; 21:186-91) carcinomas.The latest evidence that prostate basal cells expressing high levels ofTrop-2 are enriched for in vitro and in vivo stem-like activity isparticularly noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008;105:20882-7).

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue (Stein et al., 1990). Trop-2 is expressedprimarily by carcinomas such as carcinomas of the lung, stomach, urinarybladder, breast, ovary, uterus, and prostate. Localization and therapystudies using radiolabeled murine RS7 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Stein et al.,1990; Stein et al., 1991).

Strong RS7 staining has been demonstrated in tumors from the lung,breast, bladder, ovary, uterus, stomach, and prostate. (Stein et al.,Int. J. Cancer 55:938, 1993) The lung cancer cases comprised bothsquamous cell carcinomas and adenocarcinomas. (Stein et al., Int. J.Cancer 55:938, 1993) Both cell types stained strongly, indicating thatthe RS7 antibody does not distinguish between histologic classes ofnon-small-cell carcinoma of the lung.

The RS7 MAb is rapidly internalized into target cells (Stein et al.,1993). The internalization rate constant for RS7 MAb is intermediatebetween the internalization rate constants of two other rapidlyinternalizing MAbs, which have been demonstrated to be useful forimmunotoxin production. (Id.) It is well documented that internalizationof immunotoxin conjugates is a requirement for anti-tumor activity.(Pastan et al., Cell 47:641, 1986) Internalization of drugimmunoconjugates has been described as a major factor in anti-tumorefficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85: 1189, 1988) Thus,the RS7 antibody exhibits several important properties for therapeuticapplications.

While the hRS7 antibody is preferred, other anti-Trop-2 antibodies areknown and/or publicly available and in alternative embodiments may beutilized in the subject ADCs. While humanized or human antibodies arepreferred for reduced immunogenicity, in alternative embodiments achimeric antibody may be of use. As discussed below, methods of antibodyhumanization are well known in the art and may be utilized to convert anavailable murine or chimeric antibody into a humanized form.

Anti-Trop-2 antibodies are commercially available from a number ofsources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417(LifeSpan BioSciences, Inc., Seattle, Wash.); 10428-MM01, 10428-MM02,10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54(eBioscience, San Diego, Calif.); sc-376181, sc-376746, Santa CruzBiotechnology (Santa Cruz, Calif.); MM0588-49D6, (Novus Biologicals,Littleton, Colo.); ab79976, and ab89928 (ABCAM®, Cambridge, Mass.).

Other anti-Trop-2 antibodies have been disclosed in the patentliterature. For example, U.S. Publ. No. 2013/0089872 disclosesanti-Trop-2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107(Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM BP-11253),T6-16 (Accession No. FERM BP-11346), and T5-86 (Accession No. FERMBP-11254), deposited with the International Patent Organism Depositary,Tsukuba, Japan. U.S. Pat. No. 5,840,854 disclosed the anti-Trop-2monoclonal antibody BR110 (ATCC No. HB11698). U.S. Pat. No. 7,420,040disclosed an anti-Trop-2 antibody produced by hybridoma cell lineAR47A6.4.2, deposited with the IDAC (International Depository Authorityof Canada, Winnipeg, Canada) as accession number 141205-05. U.S. Pat.No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridomacell line AR52A301.5, deposited with the IDAC as accession number141205-03. U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representativeantibody were deposited with the American Type Culture Collection(ATCC), Accession Nos. PTA-12871 and PTA-12872. U.S. Pat. No. 8,715,662discloses anti-Trop-2 antibodies produced by hybridomas deposited at theAID-ICLC (Genoa, Italy) with deposit numbers PD 08019, PD 08020 and PD08021. U.S. Patent Application Publ. No. 20120237518 disclosesanti-Trop-2 antibodies 77220, KM4097 and KM4590. U.S. Pat. No. 8,309,094(Wyeth) discloses antibodies A1 and A3, identified by sequence listing.The Examples section of each patent or patent application cited above inthis paragraph is incorporated herein by reference. Non-patentpublication Lipinski et al. (1981, Proc Natl. Acad Sci USA, 78:5147-50)disclosed anti-Trop-2 antibodies 162-25.3 and 162-46.2.

Numerous anti-Trop-2 antibodies are known in the art and/or publiclyavailable. As discussed below, methods for preparing antibodies againstknown antigens were routine in the art. The sequence of the human Trop-2protein was also known in the art (see, e.g., GenBank Accession No.CAA54801.1). Methods for producing humanized, human or chimericantibodies were also known. The person of ordinary skill, reading theinstant disclosure in light of general knowledge in the art, would havebeen able to make and use the genus of anti-Trop-2 antibodies in thesubject ADCs.

Anti-CD3 Antibodies

A variety of antibodies against CD3 that may be used in the claimedmethods and compositions are publicly known and/or commerciallyavailable, such as from LSBio (catalog Nos. LS-B6698, LS-B8669;LS-B8765, LS-C96311, LS-058677, etc.); ABCAM® (catalog Nos. ab5690,ab16669, ab699, ab828, ab8671, etc.); Santa Cruz Biotechnology (catalogNos.sc-20047, sc-20080, sc-19590, sc-59008, sc-101442, etc.); and manyother suppliers.

In a preferred embodiment, the amino acid sequence of the anti-CD3moiety, used as part of a DNL® complex, is as disclosed below in SEQ IDNO:96 to SEQ ID NO:101. However, the person of ordinary skill willrealize that any known anti-CD3 antibody may be utilized in the claimedmethods and compositions. Preferably, the antibody moieties of use arehumanized or human.

Leukocyte Redirecting Bispecific Antibody Complexes

In preferred embodiments, the subject bispecific antibodies comprise ananti-CD3×anti-Trop-2 antibody. As discussed above, various antibodiesagainst CD3 or Trop-2 are known in the art and any such known antibodymay be utilized. However, in alternative embodiments, antibodies againstother leukocyte antigens than CD3 or against other disease-associatedantigens than Trop-2 may be utilized.

Exemplary T-cell antigens include CD2, CD3, CD4, CD5, CD6, CD8, CD25,CD28, CD30, CD40, CD40L, CD44, CD45, CD69 and CD90. Other exemplaryantigens may be selected from CD8, CD16, CD56, CD57, ADAM17, and CD137for NK cells; CD74, HLA-DR alpha chain, CD14, CD16, CD64 and CD89 formonocytes; and CEACAM6, CEACAM8, CD16b, CD32a, CD89, CD177, CD11a, CD11band SLC44A2 for neutrophils. Preferably, the anti-T-cell antibody bindsto CD3, or the anti-NK antibody binds to CD16. As discussed below, manyexamples of disease-associated antigens, such as tumor-associatedantigens (TAAs) are known. An exemplary preferred TAA is Trop-2.

Certain alternative embodiments may concern an anti-CD3×anti-CD19bispecific antibody. Various bispecific anti-CD3×anti-CD19 antibodiesare known in the art and presently in clinical development, such asBITE® (Bispecific T-cell Engager) (e.g., Nagorsen et al., 2009, Leukemia& Lymphoma 50:886-91; Amann et al., 2009, J Immunother 32:453-64;Baeuerle and Reinhardt, 2009, Cancer Res 69:4941-44) and DART® (see,e.g., Moore et al., 2011, Blood 117:4542-51; Veri et al., 2010,Arthritis Rheum 62:1933-43). Blinatumomab is a BITE® antibody comprisingV_(H) and V_(L) domains of anti-CD3 and anti-CD19 antibody fragments,connected with a 5-amino acid linker and expressed as a singlepolypeptide chain that anneals to itself to form the antigen-bindingsites. It is thought that blinatumomab acts by bringing theT-cell-specific CD3 and B-cell specific CD19 antigens into closeproximity, to initiate a T-cell cytotoxic response against thejuxtaposed B cells, which does not require T-cell specificity to thecancer cells (e.g., Portell et al., 2013, Clin Pharmacol 5 (Suppl 1):5-11). Due to its short half-life, blinatumomab requires continuousintravenous infusion to be effective, (Portell et al., 2013). A phase IItrial of B-cell ALL patients with persistent or relapsed minimalresidual disease reported an approximately 80% rate of completeremission (Portell et al., 2013).

Doses of blinatumomab as low as 0.005 mg/m²/day were reported to beeffective to eliminate cancer cells in non-Hodgkin's lymphoma patients(Bargou et al., 2008, Science 321:974-77). Partial and completeremissions were observed starting at a dose level of 0.015 mg and allsix patients tested at a dose of 0.06 mg experienced a tumor regression(Bargou et al., 2008). In vitro, blinatumomab induced 50% cell lysis ofMEC-1 cells at a concentration of 10 pg/mL (Topp et al., 2012, Blood120:5185-87; Bassan et al., 2012, Blood 120:5094-95).

The anti-CD19 portion of blinatumomab was derived from the HD37hybridoma (see, e.g., U.S. Pat. No. 7,575,923, the Examples section ofwhich is incorporated herein by reference), which is publicly available(e.g., Santa Cruz Biotechnology Cat. No. sc-18894). The anti-CD3 portionof blinatumomab was derived from the TR66 hybridoma (U.S. Pat. No.7,575,923; Traunecker et al., 1991, EMBO J. 10:3655-59), also publiclyavailable (e.g., Enzo Life Sciences, catalog No. ALX-804-822-C100).

A variety of antibodies against CD19 are publicly known and/orcommercially available, such as from Santa Cruz Biotechnology (catalogNos. sc-390244, sc-373897, sc-18894, sc-18896, etc.); ABCAM® (catalogNos. ab25232, ab134114, ab140981, ab1255, etc.); ABBIOTEC™ (catalog Nos.252262, 252248, 250585, 251063, etc.) and many other vendors.

In a preferred embodiment, the anti-CD19 antibody moiety is a humanizedA19 antibody, comprising the light chain CDR sequences CDR1KASQSVDYDGDSYLN (SEQ ID NO:90); CDR2 DASNLVS (SEQ ID NO:91); and CDR3QQSTEDPWT (SEQ ID NO:92) and the heavy chain CDR sequences CDR1 SYWMN(SEQ ID NO:93); CDR2 QIWPGDGDTNYNGKFKG (SEQ ID NO:94) and CDR3RETTTVGRYYYAMDY (SEQ ID NO:95).

Other anti-CD3×anti-CD19 bispecific antibodies are known, such as DART®,which also incorporates the anti-CD19 Fv sequences of HD37 and theanti-CD3 Fv sequences of TR66 (Moore et al., 2011, Blood 117:4542-51;Veri et al., 2010, Arthritis Rheum 62:1933-43). Moore et al. (2011)reported that DART® bispecific antibodies were more potent at inducing Bcell lysis than single-chain, bispecific antibodies (BITE®) bearingidentical anti-CD19 and anti-CD3 variable region sequences, with EC₅₀values in the pg/mL range (Moore et al., 2011). Other anti-CD3×anti-CD19bispecific antibodies besides DART® and BITE® have been reported (see,e.g., Wei et al., 2012, Cell Oncol 35:423-34; Portner et al., 2012,Cancer Immunol Immunother 61:1869-75; Zhou et al., 2012, BiotechnolLett. 34:1183-91). In certain embodiments, any known anti-CD3×anti-CD19bispecific antibody may be used to induce an immune response againstdisease-associated cells.

Catumaxomab is an anti-CD3×anti-EpCAM bispecific antibody that has beenapproved in Europe for treatment of malignant ascites associated withmetastasizing cancer (Chames & Baty, 2009, MAbs 1:539-47). In a mousemodel system, catumaxomab was able to kill tumor cells at aconcentration range of 10 pM and was reported to lead to totaleradication of melanoma tumors (Chames & Baty, 2009). Human clinicaltrials with ovarian cancer patients with malignant ascites also showed astatistically significant efficacy (Chames & Baty, 2009). However, thehigh immunogenicity of the rat/mouse hybrid bsAb may limit i.v.administration of the antibody (Chames & Baty, 2009). The use ofanti-tumor bsAbs is not limited to anti-CD3×anti-CD19, but has alsoincluded anti-HER2×anti-CD64 (MDX-210, MDX-H210), anti-EGFR×anti-CD64(MDX-447), anti-CD30×anti-CD16 (HRS-3/A9), anti-HER2×anti-CD3 (Her2Bi),anti-CD20×anti-CD3 (CD20Bi, Bi20), anti-EpCAM×anti-CD3 (catumaxomab,MT110), anti-HER2×anti-CD3 (ertumaxomab), and anti-NG2×anti-CD28 (rM28)(Chames & Baty, 2009).

The person of ordinary skill will realize that the subject leukocyteredirecting bispecific antibodies are not limited toanti-CD3×anti-Trop-2 constructs, but may comprise antibodies against anyknown disease-associated antigens attached to an anti-CD3 antibodymoiety. Alternatively, antibodies against other T-cell antigens besidesCD3, or other antigens expressed on NK cells, monocytes or neutrophilsmay also be used. Exemplary T-cell antigens include, but are not limitedto, CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44,CD45, CD69 and CD90. Other exemplary antigens may be selected from CD8,CD16, CD56, CD57, ADAM17, KIR and CD137 for NK cells; CD74, HLA-DR alphachain, CD14, CD16, CD64 and CD89 for monocytes; and CEACAM6, CEACAM8,CD16b, CD32a, CD89, CD177, CD11a, CD11b and SLC44A2 for neutrophils.Antibodies against each of the leukocyte antigens are publicly knownand/or publicly available (see, e.g., ABCAM® catalog numbers ab131276,ab139266, ab8360, ab51312, ab846, ab133616, ab75877, ab133255, ab109217,ab93278, ab17147, ab115851, ab128955, ab13463, ab85986; Santa CruzBiotechnology catalog numbers sc-46683, sc-59047; Enzo Life Sciences,Inc. catalog number ALX-805-037-C100; Sino Biological Inc. catalognumbers 12211-RP02, 11150-R074; Millipore catalog numbers 04-1102,04-1102, MAB1406). These and numerous other anti-leukocyte antibodieswere publicly available and could have been used in the subjectleukocyte redirecting bsAbs. As discussed below, numerous antibodiesagainst a wide variety of disease-associated antigens were publiclyknown and/or commercially available and could have been used in thesubject leukocyte redirecting bispecific antibodies. Other exemplaryleukocyte redirecting bsAbs of potential use include FBTA05(anti-CD20×anti-CD3) and TRBS07 (anti-GD2×anti-CD3).

Interferon Therapy

In various embodiments, the subject bispecific antibodies may be used incombination with one or more interferons, such as interferon-α,interferon-β or interferon-λ, preferably interferon-α. Human interferonsare well known in the art and the amino acid sequences of humaninterferons may be readily obtained from public databases (e.g., GenBankAccession Nos. AAA52716.1; AAA52724; AAC41702.1; EAW56871.1; EAW56870.1;EAW56869.1). Human interferons may also be commercially obtained from avariety of vendors (e.g., Cell Signaling Technology, Inc., Danvers,Mass.; Genentech, South San Francisco, Calif.; EMD Millipore, Billerica,Mass.).

Interferon-α (IFNα) has been reported to have anti-tumor activity inanimal models of cancer (Ferrantini et al., 1994, J Immunol 153:4604-15)and human cancer patients (Gutterman et al., 1980, Ann Intern Med93:399-406). IFNα can exert a variety of direct anti-tumor effects,including down-regulation of oncogenes, up-regulation of tumorsuppressors, enhancement of immune recognition via increased expressionof tumor surface MHC class I proteins, potentiation of apoptosis, andsensitization to chemotherapeutic agents (Gutterman et al., 1994, PNASUSA 91:1198-205; Matarrese et al., 2002, Am J Pathol 160:1507-20;Mecchia et al., 2000, Gene Ther 7:167-79; Sabaawy et al., 1999, Int JOncol 14:1143-51; Takaoka et al, 2003, Nature 424:516-23). For sometumors, IFNα can have a direct and potent anti-proliferative effectthrough activation of STAT1 (Grimley et al., 1998 Blood 91:3017-27).Interferon-α2b has been conjugated to anti-tumor antibodies, such as thehL243 anti-HLA-DR antibody and depletes lymphoma and myeloma cells invitro and in vivo (Rossi et al., 2011, Blood 118:1877-84).

Indirectly, IFNα can inhibit angiogenesis (Sidky and Borden, 1987,Cancer Res 47:5155-61) and stimulate host immune cells, which may bevital to the overall antitumor response but has been largelyunder-appreciated (Belardelli et al., 1996, Immunol Today 17:369-72).IFNα has a pleiotropic influence on immune responses through effects onmyeloid cells (Raefsky et al, 1985, J Immunol 135:2507-12; Luft et al,1998, J Immunol 161:1947-53), T-cells (Carrero et al, 2006, J Exp Med203:933-40; Pilling et al., 1999, Eur J Immunol 29:1041-50), and B-cells(Le et al, 2001, Immunity 14:461-70). As an important modulator of theinnate immune system, IFNα induces the rapid differentiation andactivation of dendritic cells (Belardelli et al, 2004, Cancer Res64:6827-30; Paquette et al., 1998, J Leukoc Biol 64:358-67; Santini etal., 2000, J Exp Med 191:1777-88) and enhances the cytotoxicity,migration, cytokine production and antibody-dependent cellularcytotoxicity (ADCC) of NK cells (Biron et al., 1999, Ann Rev Immunol17:189-220; Brunda et al. 1984, Cancer Res 44:597-601).

Interferon-β has been reported to be efficacious for therapy of avariety of solid tumors. Patients treated with 6 million units of IFN-βtwice a week for 36 months showed a decreased recurrence ofhepatocellular carcinoma after complete resection or ablation of theprimary tumor in patients with HCV-related liver cancer (Ikeda et al.,2000, Hepatology 32:228-32). Gene therapy with interferon-β inducedapoptosis of glioma, melanoma and renal cell carcinoma (Yoshida et al.,2004, Cancer Sci 95:858-65). Endogenous IFN-β has been observed toinhibit tumor growth by inhibiting angiogenesis in vivo (Jablonska etal., 2010, J Clin Invest. 120:1151-64.)

IFN-λs, designated as type III interferons, are a newly described groupof cytokines that consist of IFN-λ1, 2, 3 (also referred to asinterleukin-29, 28A, and 28B, respectively), that are geneticallyencoded by three different genes located on chromosome 19 (Kotenko etal., 2003, Nat Immunol 4:69-77; Sheppard et al., 2003, Nat Immunol4:63-8). At the protein level, IFN-λ2 and -λ3 are is highly homologous,with 96% amino acid identity, while IFN-λ1 shares approximately 81%homology with IFN-λ2 and -λ3 (Sheppard et al., 2003, Nat Immunol4:63-8). IFN-λs activate signal transduction via the JAK/STAT pathwaysimilar to that induced by type I IFN, including the activation of JAK1and TYK2 kinases, the phosphorylation of STAT proteins, and theactivation of the transcription complex of IFN-stimulated gene factor 3(ISGF3) (Witte et al., 2010, Cytokine Growth Factor Rev 21:237-51; Zhouet al., 2007, J Virol 81:7749-58).

A major difference between type III and type I IFN systems is thedistribution of their respective receptor complexes. IFN-α/β signalsthrough two extensively expressed type I interferon receptors, and theresulting systemic toxicity associated with IFN-α/β administration haslimited their use as therapeutic agents (Pestka et al., 2007, J BiolChem 282:20047-51). In contrast, IFN-λs signal through a heterodimericreceptor complex consisting of unique IFN-λ receptor 1 (IFN-λR1) andIL-10 receptor 2 (IL-10R2). As previously reported (Witte et al., 2009,Genes Immun 10:702-14), IFN-λR1 has a very restricted expression patternwith the highest levels in epithelial cells, melanocytes, andhepatocytes, and the lowest level in primary central nervous system(CNS) cells. Blood immune system cells express high levels of a shortIFN-λ receptor splice variant (sIFN-λR1) that inhibits IFN-λ action. Thelimited responsiveness of neuronal cells and immune cells implies thatthe severe toxicity frequently associated with IFN-α therapy may beabsent or significantly reduced with IFN-λs (Witte et al., 2009, GenesImmun 10:702-14; Witte et al., 2010, Cytokine Growth Factor Rev21:237-51). A recent publication reported that while IFN-α and IFN-λinduce expression of a common set of ISGs (interferon-stimulated genes)in hepatocytes, unlike IFN-α, administration of IFN-λ did not induceSTAT activation or ISG expression in purified lymphocytes or monocytes(Dickensheets et al., 2013, J Leukoc Biol. 93, published online Dec. 20,2012). It was suggested that IFN-λ may be superior to IFN-α fortreatment of chronic HCV infection, as it is less likely to induceleukopenias that are often associated with IFN-α therapy (Dickensheetset al., 2013).

IFN-λs display structural features similar to IL-10-related cytokines,but functionally possess type I IFN-like anti-viral andanti-proliferative activity (Witte et al., 2009, Genes Immun 10:702-14;Ank et al., 2006, J Virol 80:4501-9; Robek et al., 2005, J Virol79:3851-4). IFN-λ1 and -λ2 have been demonstrated to reduce viralreplication or the cytopathic effect of various viruses, including DNAviruses (hepatitis B virus (Robek et al., 2005, J Virol 79:3851-4, Doyleet al., 2006, Hepatology 44:896-906) and herpes simplex virus 2 (Ank etal., 2008, J Immunol 180:2474-85)), ss (+) RNA viruses (EMCV; Sheppardet al., 2003, Nat Immunol 4:63-8) and hepatitis C virus (Robek et al.,2005, J Virol 79:3851-4, Doyle et al., 2006, Hepatology 44:896-906;Marcello et al., 2006, Gastroenterol 131:1887-98; Pagliaccetti et al.,2008, J Biol Chem 283:30079-89), ss (−) RNA viruses (vesicularstomatitis virus; Pagliaccetti et al., 2008, J Biol Chem 283:30079-89)and influenza-A virus (Jewell et al., 2010, J Virol 84:11515-22) anddouble-stranded RNA viruses, such as rotavirus (Pott et al., 2011, PNASUSA 108:7944049). IFN-λ3 has been identified from genetic studies as akey cytokine in HCV infection (Ge et al., 2009, Nature 461:399-401), andhas also shown potent activity against EMCV (Dellgren et al., 2009,Genes Immun 10:125-31). A deficiency of rhinovirus-induced IFN-λproduction was reported to be highly correlated with the severity ofrhinovirus-induced asthma exacerbation (Contoli et al., 2006, Nature Med12:1023-26) and IFN-λ therapy has been suggested as a new approach fortreatment of allergic asthma (Edwards and Johnston, 2011, EMBO Mol Med3:306-8; Koltsida et al., 2011, EMBO Mol Med 3:348-61).

The anti-proliferative activity of IFN-λs has been established inseveral human cancer cell lines, including neuroendocrine carcinoma BON1(Zitzmann et al., 2006, Biochem Biophys Res Commun 344:1334-41),glioblastoma LN319 (Meager et al., 2005, Cytokine 31:109-18),immortalized keratinocyte HaCaT (Maher et al., 2008, Cancer Biol Ther7:1109-15), melanoma F01 (Guenterberg et al., 2010, Mol Cancer Ther9:510-20), and esophageal carcinoma TE-11 (Li et al., 2010, Eur J Cancer46:180-90). In animal models, IFN-λs induce both tumor apoptosis anddestruction through innate and adaptive immune responses, suggestingthat local delivery of IFN-λ might be a useful adjunctive strategy inthe treatment of human malignancies (Numasaki et al., 2007, J Immunol178:5086-98). A Fab-linked interferon-λ was demonstrated to have potentanti-tumor and anti-viral activity in targeted cells (Liu et al., 2013,PLoS One 8:e63940).

In clinical settings, PEGylated IFN-λ1 (PEG-IFN-λ1) has beenprovisionally used for patients with chronic hepatitis C virusinfection. In a phase Ib study (n=56), antiviral activity was observedat all dose levels (0.5-3.0 μg/kg), and viral load reduced 2.3 to 4.0logs when PEG-IFN-λ1 was administrated to genotype 1 HCV patients whorelapsed after IFN-α therapy (Muir et al., 2010, Hepatology 52:822-32).A phase IIb study (n=526) showed that patients with HCV genotypes 1 and4 had significantly higher response rates to treatment with PEG-IFN-λ1compared to PEG-IFN-α. At the same time, rates of adverse eventscommonly associated with type I interferon treatment were lower withPEG-IFN-λ1 than with PEG-IFN-α. Neutropenia and thrombocytopenia wereinfrequently observed and the rates of flu-like symptoms, anemia, andmusculoskeletal symptoms decreased to about ⅓ of that seen withPEG-IFN-α treatment. However, rates of serious adverse events,depression and other common adverse events (≥10%) were similar betweenPEG-IFN-λ1 and PEG-IFN-α. Higher rates of hepatotoxicity were seen inthe highest-dose PEG-IFN-λ1 compared with PEG-IFN-α (“InvestigationalCompound PEG-Interferon Lambda Achieved Higher Response Rates with FewerFlu-like and Musculoskeletal Symptoms and Cytopenias Than PEG-InterferonAlfa in Phase IIb Study of 526 Treatment-Naive Hepatitis C Patients,”Apr. 2, 2011, Press Release from Bristol-Myers Squibb).

In various embodiments, the subject leukocyte redirecting bispecificantibodies, ADCs and/or checkpoint inhibitor mAbs may be used incombination with one or more interferons, such as interferon-α,interferon-β, interferon-λ1, interferon-λ2, or interferon-λ3. When usedwith other agents, the interferon may be administered prior to,concurrently with, or after the other agent. When administeredconcurrently, the interferon may be either conjugated to or separatefrom the other agent.

Checkpoint Inhibitor Antibodies

Studies with checkpoint inhibitor antibodies for cancer therapy havegenerated unprecedented response rates in cancers previously thought tobe resistant to cancer treatment (see, e.g., Ott & Bhardwaj, 2013,Frontiers in Immunology 4:346; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85; Pardoll, 2012, Nature Reviews Cancer 12:252-64; Mavilio &Lugli,). Therapy with antagonistic checkpoint blocking antibodiesagainst immune system checkpoints such as CTLA4, PD1 and PD-L1 are oneof the most promising new avenues of immunotherapy for cancer and otherdiseases. In contrast to the majority of anti-cancer agents, checkpointinhibitors do not target tumor cells directly, but rather targetlymphocyte receptors or their ligands in order to enhance the endogenousantitumor activity of the immune system. (Pardoll, 2012, Nature ReviewsCancer 12:252-264) Because such antibodies act primarily by regulatingthe immune response to diseased cells, tissues or pathogens, they may beused in combination with other therapeutic modalities, such as thesubject leukocyte redirecting bispecific antibodies, ADCs and/orinterferons to enhance the anti-tumor effect of such agents.

It is now clear that tumors can escape immune surveillance by co-optingcertain immune-checkpoint pathways, particularly in T cells that arespecific for tumor antigens (Pardoll, 2012, Nature Reviews Cancer12:252-264). Because many such immune checkpoints are initiated byligand-receptor interactions, they can be readily blocked by antibodiesagainst the ligands and/or their receptors (Pardoll, 2012, NatureReviews Cancer 12:252-264). Although checkpoint inhibitor antibodiesagainst CTLA4, PD1 and PD-L1 are the most clinically advanced, otherpotential checkpoint antigens are known and may be used as the target oftherapeutic antibodies, such as LAG3, B7-H3, B7-H4 and TIM3 (Pardoll,2012, Nature Reviews Cancer 12:252-264).

Programmed cell death protein 1 (PD1, also known as CD279) encodes acell surface membrane protein of the immunoglobulin superfamily, whichis expressed in B cells and NK cells (Shinohara et al., 1995, Genomics23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45;Finger et al., 1997, Gene 197:177-87; Pardoll, 2012, Nature ReviewsCancer 12:252-264). The major role of PD1 is to limit the activity of Tcells in peripheral tissues during inflammation in response toinfection, as well as to limit autoimmunity (Pardoll, 2012, NatureReviews Cancer 12:252-264). PD1 expression is induced in activated Tcells and binding of PD1 to one of its endogenous ligants acts toinhibit T-cell activation by inhibiting stimulatory kinases (Pardoll,2012, Nature Reviews Cancer 12:252-264). PD1 also acts to inhibit theTCR “stop signal” (Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1is highly expressed on T_(reg) cells and may increase theirproliferation in the presence of ligand (Pardoll, 2012, Nature ReviewsCancer 12:252-264).

Anti-PD1 antibodies have been used for treatment of melanoma,non-small-cell lung cancer, bladder cancer, prostate cancer, colorectalcancer, head and neck cancer, triple-negative breast cancer, leukemia,lymphoma and renal cell cancer (Topalian et al., 2012, N Engl J Med366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger etal., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013,Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85). Because PD1/PD-L1 and CTLA4 act by different pathways, it ispossible that combination therapy with checkpoint inhibitor antibodiesagainst each may provide an enhanced immune response.

Exemplary anti-PD1 antibodies include lambrolizumab (MK-3475, MERCK),nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), andpidilizumab (CT-011, CURETECH LTD.). Anti-PD1 antibodies arecommercially available, for example from ABCAM® (AB137132), BIOLEGEND®(EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).

Programmed cell death 1 ligand 1 (PD-L1, also known as CD274 and B7-H1)is a ligand for PD1, found on activated T cells, B cells, myeloid cellsand macrophages. Although there are two endogenous ligands for PD1—PD-L1and PD-L2, anti-tumor therapies have focused on anti-PD-L1 antibodies.The complex of PD1 and PD-L1 inhibits proliferation of CD8+ T cells andreduces the immune response (Topalian et al., 2012, N Engl J Med366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65). Anti-PD-L1antibodies have been used for treatment of non-small cell lung cancer,melanoma, colorectal cancer, renal-cell cancer, pancreatic cancer,gastric cancer, ovarian cancer, breast cancer, and hematologicmalignancies (Brahmer et al., N Eng J Med 366:2455-65; Ott et al., 2013,Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger etal., 2008, Clin Cancer Res 14:13044-51).

Exemplary anti-PD-L1 antibodies include MDX-1105 (MEDAREX), MEDI4736(MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB).Anti-PD-L1 antibodies are also commercially available, for example fromAFFYMETRIX EBIOSCIENCE (MIH1).

Cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152) is also amember of the immunoglobulin superfamily that is expressed exclusivelyon T-cells. CTLA4 acts to inhibit T-cell activation and is reported toinhibit helper T-cell activity and enhance regulatory T-cellimmunosuppressive activity (Pardoll, 2012, Nature Reviews Cancer12:252-264). Although the precise mechanism of action of CTL4-A remainsunder investigation, it has been suggested that it inhibits T cellactivation by outcompeting CD28 in binding to CD80 and CD86, as well asactively delivering inhibitor signals to the T cell (Pardoll, 2012,Nature Reviews Cancer 12:252-264). Anti-CTL4A antibodies have been usedin clinical trials for treatment of melanoma, prostate cancer, smallcell lung cancer, non-small cell lung cancer (Robert & Ghiringhelli,2009, Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19:5300;Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med11:89). A significant feature of anti-CTL4A is the kinetics ofanti-tumor effect, with a lag period of up to 6 months after initialtreatment required for physiologic response (Pardoll, 2012, NatureReviews Cancer 12:252-264). In some cases, tumors may actually increasein size after treatment initiation, before a reduction is seen (Pardoll,2012, Nature Reviews Cancer 12:252-264).

Exemplary anti-CTLA4 antibodies include ipilimumab (Bristol-MyersSquibb) and tremelimumab (PFIZER). Anti-PD1 antibodies are commerciallyavailable, for example from ABCAM® (AB134090), SINO BIOLOGICAL INC.(11159-H03H, 11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572,PA5-23967, PA5-26465, MA1-12205, MA1-35914). Ipilimumab has recentlyreceived FDA approval for treatment of metastatic melanoma (Wada et al.,2013, J Transl Med 11:89).

The person of ordinary skill will realize that methods of determiningoptimal dosages of checkpoint inhibitor antibodies to administer to apatient in need thereof, either alone or in combination with one or moreother agents, may be determined by standard dose-response and toxicitystudies that are well known in the art. In an exemplary embodiment, acheckpoint inhibitor antibody may preferably be administered at about0.3-10 mg/kg, or the maximum tolerated dose, administered about everythree weeks or about every six weeks. Alternatively, the checkpointinhibitor antibody may be administered by an escalating dosage regimenincluding administering a first dosage at about 3 mg/kg, a second dosageat about 5 mg/kg, and a third dosage at about 9 mg/kg. Alternatively,the escalating dosage regimen includes administering a first dosage ofcheckpoint inhibitor antibody at about 5 mg/kg and a second dosage atabout 9 mg/kg. Another stepwise escalating dosage regimen may includeadministering a first dosage of checkpoint inhibitor antibody about 3mg/kg, a second dosage of about 3 mg/kg, a third dosage of about 5mg/kg, a fourth dosage of about 5 mg/kg, and a fifth dosage of about 9mg/kg. In another aspect, a stepwise escalating dosage regimen mayinclude administering a first dosage of 5 mg/kg, a second dosage of 5mg/kg, and a third dosage of 9 mg/kg. Exemplary reported dosages ofcheckpoint inhibitor mAbs include 3 mg/kg ipilimumab administered everythree weeks for four doses; 10 mg/kg ipilimumab every three weeks foreight cycles; 10 mg/kg every three weeks for four cycles then every 12weeks for a total of three years; 10 mg/kg MK-3475 every two or everythree weeks; 2 mg/kg MK-3475 every three weeks; 15 mg/kg tremilimumabevery three months; 0.1, 0.3, 1, 3 or 10 mg/kg nivolumab every two weeksfor up to 96 weeks; 0.3, 1, 3, or 10 mg/kg BMS-936559 every two weeksfor up to 96 weeks (Kyi & Postow, Oct. 23, 2013, FEBS Lett [Epub aheadof print]; Callahan & Wolchok, 2013, J Leukoc Biol 94:41-53).

These and other known agents that stimulate immune response to tumorsand/or pathogens may be used in combination with leukocyte redirectingbispecific antibodies alone or in further combination with aninterferon, such as interferon-α, and/or an antibody-drug conjugate forimproved cancer therapy. Other known co-stimulatory pathway modulatorsthat may be used in combination include, but are not limited to,agatolimod, belatacept, blinatumomab, CD40 ligand, anti-B7-1 antibody,anti-B7-2 antibody, anti-B7-H4 antibody, AG4263, eritoran, anti-OX40antibody, ISF-154, and SGN-70; B7-1, B7-2, ICAM-1, ICAM-2, ICAM-3, CD48,LFA-3, CD30 ligand, CD40 ligand, heat stable antigen, B7h, OX40 ligand,LIGHT, CD70 and CD24.

In certain embodiments, anti-KIR antibodies may also be used incombination with leukocyte-redirecting bsAbs, interferons, ADCs and/orcheckpoint inhibitor antibodies. NK cells mediate anti-tumor andanti-infectious agent activity by spontaneous cytotoxicity and by ADCCwhen activated by antibodies (Kohrt et al., 2013, Blood, [Epub ahead ofprint Dec. 10, 2013]). The degree of cytotoxic response is determined bya balance of inhibitory and activating signals received by the NK cells(Kohrt et al., 2013). The killer cell immunoglobulin-like receptor (KIR)mediates an inhibitory signal that decreases NK cell response. Anti-KIRantibodies, such as lirlumab (Innate Pharma) and IPH2101 (Innate Pharma)have demonstrated anti-tumor activity in multiple myeloma (Benson etal., 2012, Blood 120:4324-33). In vitro, anti-KIR antibodies prevent thetolerogenic interaction of NK cells with target cells and augments theNK cell cytotoxic response to tumor cells (Kohrt et al., 2013). In vivo,in combination with rituximab (anti-CD20), anti-KIR antibodies at a doseof 0.5 mg/kg induced enhanced NK cell-mediated, rituximab-dependentcytotoxicity against lymphoma tumors (Kohrt et al., 2013). Anti-KIR mAbsmay be combined with ADCs, leukocyte-redirecting bsAbs, interferonsand/or checkpoint inhibitor antibodies to potentiate cytotoxicity totumor cells or pathogenic organisms.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: PHAGE DISPLAY LABORATORY MANUAL, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23)from Abgenix (Fremont, Calif.). In the XENOMOUSE® and similar animals,the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding V_(κ) (variable light chain) and V_(H) (variableheavy chain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of an antibody from a cell thatexpresses a murine antibody can be cloned by PCR amplification andsequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci. USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized antibody can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine antibody by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The V_(κ) sequence for the antibody may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for V_(κ) can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the V_(κ) andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human antibody. Alternatively, the V_(κ) andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, scFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. Nos. 4,704,692; 4,946,778; Raag and Whitlow,FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9: 132-137 (1991).

Techniques for producing single domain antibodies (DABs or VHH) are alsoknown in the art, as disclosed for example in Cossins et al. (2006, ProtExpress Purif 51:253-259), incorporated herein by reference. Singledomain antibodies may be obtained, for example, from camels, alpacas orllamas by standard immunization techniques. (See, e.g., Muyldermans etal., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75,2003; Maass et al., J Immunol Methods 324:13-25, 2007). The VHH may havepotent antigen-binding capacity and can interact with novel epitopesthat are inacessible to conventional VH-VL pairs. (Muyldermans et al.,2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgGantibodies (HCAbs) (Maass et al., 2007). Alpacas may be immunized withknown antigens, such as TNF-α, and VHHs can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca VHH coding sequences have been identifiedand may be used to construct alpaca VHH phage display libraries, whichcan be used for antibody fragment isolation by standard biopanningtechniques well known in the art (Maass et al., 2007). In certainembodiments, anti-pancreatic cancer VHH antibody fragments may beutilized in the claimed compositions and methods.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Stickler et al.,2011). It has been reported that G1m1 antibodies contain allotypicsequences that tend to induce an immune response when administered tonon-G1m1 (nG1m1) recipients, such as G1m3 patients (Stickler et al.,2011). Non-G1m1 allotype antibodies are not as immunogenic whenadministered to G1m1 patients (Stickler et al., 2011).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:85) and veltuzumab (SEQ IDNO:86).

Rituximab heavy chain variable region sequence (SEQ ID NO: 85)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 86) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Known Antibodies

Target Antigens and Exemplary Antibodies

In a preferred embodiment, antibodies are used that recognize and/orbind to antigens that are expressed at high levels on target cells andthat are expressed predominantly or exclusively on diseased cells versusnormal tissues. Exemplary antibodies of use for therapy of, for example,cancer include but are not limited to LL1 (anti-CD74), LL2 or RFB4(anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20),obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD1), nivolumab(anti-PD1), MK-3475 (anti-PD1), AMP-224 (anti-PD1), pidilizumab(anti-PD1), MDX-1105 (anti-PD-L1), MEDI4736 (anti-PD-L1), MPDL3280A(anti-PD-L1), BMS-936559 (anti-PD-L1), ipilimumab (anti-CTLA4),trevilizumab (anti-CTL4A), RS7 (anti-epithelial glycoprotein-1 (EGP-1,also known as TROP-2)), PAM4 or KC4 (both anti-mucin), MN-14(anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM5),MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu31 (an anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19),TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specificmembrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA),G250 (an anti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab(anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR);tositumomab (anti-CD20); PAM4 (aka clivatuzumab, anti-mucin), BWA-3(anti-histone H2A/H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1),PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), LG2-2 (anti-histoneH2B), and trastuzumab (anti-ErbB2). Such antibodies are known in the art(e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. PatentApplication Publ. No. 20050271671; 20060193865; 20060210475;20070087001; the Examples section of each incorporated herein byreference.) Specific known antibodies of use include hPAM4 (U.S. Pat.No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No.7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No.7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No.7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No.6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD S, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA4,alpha-fetoprotein (AFP), VEGF (e.g., AVASTIN®, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1 (TROP-2), EGP-2 (e.g.,17-1A), EGF receptor (ErbB1) (e.g., ERBITUX®), ErbB2, ErbB3, Factor H,FHL-1, Flt-3, folate receptor, Ga 733, GRO-β, HMGB-1, hypoxia induciblefactor (HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF),IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R,IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigen to which L243binds, CD66 antigens, i.e., CD66a-d or a combination thereof, MAGE,mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration-inhibitory factor(MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor (PlGF),PSA (prostate-specific antigen), PSMA, PAM4 antigen, PD1 receptor,NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100,tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumor necrosisantigens, tumor angiogenesis antigens, TNF-α, TRAIL receptor (R1 andR2), TROP-2, VEGFR, RANTES, T101, as well as cancer stem cell antigens,complement factors C3, C3a, C3b, C5a, C5, and an oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies forimmunotherapy, is Craig and Foon, Blood prepublished online Jan. 15,2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Penis, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

Anti-cancer antibodies have been demonstrated to bind to histones insome case. Kato et al. (1991, Hum Antibodies Hybridomas 2:94-101)reported tha the lung cancer-specific human monoclonal antibody HB4C5binds to histone H2B. Garzelli et al. (1994, Immunol Lett 39:277-82)observed that Epstein-Barr virus-transformed human B lymphocytes producenatural antibodies to histones. In certain embodiments, antibodiesagainst histones may be of use in the subject combinations. Knownanti-histone antibodies include, but are not limited to, BWA-3(anti-histone H2A/H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1),PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), and LG2-2(anti-histone H2B) (see, e.g., Monestier et al., 1991, Eur J Immunol21:1725-31; Monestier et al., 1993, Molec Immunol 30:1069-75).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54). Milatuzumab (hLL1) is an exemplaryanti-CD74 antibody of therapeutic use for treatment of MIF-mediateddiseases.

An example of a most-preferred antibody/antigen pair is LL1, ananti-CD74 MAb (invariant chain, class II-specific chaperone, Ii) (see,e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; the Examples section of eachincorporated herein by reference). The CD74 antigen is highly expressedon B-cell lymphomas (including multiple myeloma) and leukemias, certainT-cell lymphomas, melanomas, colonic, lung, and renal cancers,glioblastomas, and certain other cancers (Ong et al., Immunology98:296-302 (1999)). A review of the use of CD74 antibodies in cancer iscontained in Stein et al., Clin Cancer Res. 2007 Sep. 15; 13(18 Pt2):55565-5563s, incorporated herein by reference. The diseases that arepreferably treated with anti-CD74 antibodies include, but are notlimited to, non-Hodgkin's lymphoma, Hodgkin's disease, melanoma, lung,renal, colonic cancers, glioblastome multiforme, histiocytomas, myeloidleukemias, and multiple myeloma.

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets, including but not limitedto tumor-associated antigens, have been deposited at the ATCC and/orhave published variable region sequences and are available for use inthe claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572;856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625;6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173;6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206,6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694;6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759;6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444;6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459;6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914;6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440;5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136;5,587,459; 5,443,953, 5,525,338, the Examples section of each of whichis incorporated herein by reference. These are exemplary only and a widevariety of other antibodies and their hybridomas are known in the art.The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

In other embodiments, the antibody complexes bind to a MHC class I, MHCclass II or accessory molecule, such as CD40, CD54, CD80 or CD86. Theantibody complex also may bind to a leukocyte activation cytokine, or toa cytokine mediator, such as NF-κB.

In certain embodiments, one of the two different targets may be a cancercell receptor or cancer-associated antigen, particularly one that isselected from the group consisting of B-cell lineage antigens (CD19,CD20, CD21, CD22, CD23, etc.), VEGF, VEGFR, EGFR, carcinoembryonicantigen (CEA), placental growth factor (PlGF), tenascin, HER-2/neu,EGP-1, EGP-2, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD80,CD138, NCA66, CEACAM1, CEACAM6 (carcinoembryonic antigen-relatedcellular adhesion molecule 6), MUC1, MUC2, MUC3, MUC4, MUC16, IL-6,α-fetoprotein (AFP), A3, CA125, colon-specific antigen-p (CSAp), folatereceptor, HLA-DR, human chorionic gonadotropin (HCG), Ia, EL-2,insulin-like growth factor (IGF) and IGF receptor, KS-1, Le(y), MAGE,necrosis antigens, PAM-4, prostatic acid phosphatase (PAP), Pr1,prostate specific antigen (PSA), prostate specific membrane antigen(PSMA), S100, T101, TAC, TAG72, TRAIL receptors, and carbonic anhydraseIX.

Immunoconjugates

In certain embodiments, the antibodies or fragments thereof may beconjugated to one or more therapeutic or diagnostic agents. Thetherapeutic agents do not need to be the same but can be different, e.g.a drug and a radioisotope. For example, ¹³¹I can be incorporated into atyrosine of an antibody or fusion protein and a drug attached to anepsilon amino group of a lysine residue. Therapeutic and diagnosticagents also can be attached, for example to reduced SH groups and/or tocarbohydrate side chains. Many methods for making covalent ornon-covalent conjugates of therapeutic or diagnostic agents withantibodies or fusion proteins are known in the art and any such knownmethod may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein and used to chelate a therapeutic ordiagnostic agent, such as a radionuclide. Exemplary chelators includebut are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Methods of conjugation and use of chelating agents to attach metals orother ligands to proteins are well known in the art (see, e.g., U.S.Pat. No. 7,563,433, the Examples section of which is incorporated hereinby reference).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br for radioimaging. The samechelates, when complexed with non-radioactive metals, such as manganese,iron and gadolinium are useful for MRI. Macrocyclic chelates such asNOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F—Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. Pat. No.7,563,433, the Examples section of which is incorporated herein byreference.

In specific preferred embodiments, an immunoconjugate may be anantibody-drug conjugate (ADC). Two exemplary drugs of use in ADCproduction are SN-38 and a prodrug form of 2-pyrrolinodoxorubicine(P2PDox). Compositions and methods of production of SN-38-conjugatedADCs are disclosed, for example, in U.S. Pat. Nos. 7,999,083; 8,080,250;8,741,300; 8,759,496, the Figures and Examples section of each of whichare incorporated herein by reference. Compositions and methods ofproduction of P2PDox ADCs are disclosed, for example, in U.S. Pat. No.8,877,101, the Figures and Examples section of which are incorporatedherein by reference.

Methods of Producing Bispecific Antibodies

In various embodiments, the subject combination therapy may utilize oneor more bispecific antibodies (bsAbs), such as a leukocyte redirectingbsAb. A bispecific antibody as used herein is an antibody that containsbinding sites for two different antigens, or two different epitopes onthe same antigen. An antibody that can only bind to a single epitope ona single antigen is monospecific, regardless of the number ofantigen-binding sites on the antibody molecule.

Early attempts at bispecific antibody construction either utilizedchemical cross-linking or hybrid hybridomas or quadromas to join the twohalves of two different antibodies together (e.g., Staerz et al., 1985,Nature 314:628-31; Milstein and Cuello, Nature 1983; 305:537-540;Karpovsky et al., 1984, J Exp Med 160:1686-701). Although the techniqueswork to make bsAbs, various production problems made use of suchcomplexes difficult, such as the production of mixed populationscontaining different combinations of antigen-binding sites, difficultyin protein expression, the need to purify the bsAb of interest, lowyields, expense of production, etc.

More recent approaches have utilized genetically engineered constructsthat are capable of producing homogeneous products of single bsAbs,without the need for extensive purification to remove unwantedbyproducts. Such constructs have included tandem scFv, diabodies, tandemdiabodies, dual variable domain antibodies and heterodimerization usinga motif such as Ch1/Ck domain or DNL® (Chames & Baty, 2009, Curr OpinDrug Discov Devel 12:276-83; Chames & Baty, mAbs 1:539-47).

Triomabs is a variation on the quadroma approach that use a combinationof mouse IgG2a and rat IgG2b antibodies to preferentially produce therecombinant antibody, compared to the random pairing typically seen inrat/rat or mouse/mouse quadromas (Chames & Baty, mAbs 1:539-47). Ananti-CD3×anti-EpCAM bsAb (catumaxomab) created by this technique wasable to efficiently recruit macrophages and NK cells and to activate Tcells (Chames & Baty, mAbs 1:539-47). As discussed above, catumaxomabhas been approved in Europe for treatment of malignant ascites inpatients with EpCAM positive carcinomas (Chames & Baty, mAbs 1:539-47).Surprisingly, the recombinant bsAb was reported to induce only moderateanti-mouse and anti-rat responses in humans (Chames & Baty, mAbs1:539-47), probably due at least in part to the i.p. route ofadministration for ascites. Ertumaxomab is another triomab targetingHER2, which may be of use for metastatic breast cancer. Bi20 is anothertriomab that targets CD20. In vitro, Bi20 exibited efficient lyis of Bcells from CLL patients (Chames & Baty, mAbs 1:539-47).

BITE® refers to tandem scFvs that are joined by a short peptide linker(Chames & Baty, mAbs 1:539-47). Blinatumomab is an anti-CD19×anti-CD3BITE® with reported efficacy in hematologic cancers, such asnon-Hodgkin's lymphoma and ALL, at very low concentrations (Nagorsen etal., 2009, Leukemia & Lymphoma 50:886-91; Chames & Baty, mAbs 1:539-47;Topp et al., 2012, Blood 120:5185-87; Bargou et al., 2008, Science321:974-77). Another BITE® with specificity for EpCAM has been used ingastrointestinal, ovarian, colorectal and lung cancer (Amann et al.,2009, J Immunother 32:452-64; Chames & Baty, mAbs 1:539-47). AnotherBITE® (MEDI-565) targeted to CEACAM5 has been proposed for use inmelanoma, colorectal, lung, pancreatic, stomach, ovarian, uterine, andbreast cancers (Sanders et al., 1994, J Pathol 172:343-8). BITE® hasbeen reported to exhibit anti-tumor activity at picomolar or evenfemtomolar concentrations (Chames & Baty, mAbs 1:539-47).

Another method of bsAb formation, involving assembly of two heavy andtwo light chains derived from two different pre-existing antibodies, isbased on a knobs-into-holes approach that facilitates heterodimerformation and prevents homodimer formation (Schaefer et al., 2011, ProcNatl. Acad Sci USA 108:11187-92). The “CrossMab” technique furtherinvolves the exchange of heavy and light chain domains within the Fab ofone half of the bispecific antibody, making the two arms so differentthat light-heavy chain mispairing can not occur (Schaefer et al., 2011).The knobs-into-holes approach introduces amino acids with bulky sidechains into the CH3 domain of one heavy chain that fit intoappropriately designed cavities in the CH3 domain of the other heavychain. The combination of approaches prevents mis-match of both heavychain to heavy chain and heavy chain to light chain interactions,resulting in primarily a single product. The initial CrossMab, generatedagainst angiopoietin-2 (Ang-2) and VEGF-A, exhibited bindingcharacteristics comparable to the parent mAbs, with potentanti-angiogenic and anti-tumoral activity (Schaefer et al., 2011, ProcNatl. Acad Sci USA 108:11187-92; Kienast et al., Clin Cancer Res, Oct.25, 2013, Epub ahead of print).

In addition to the DART™ technology discussed above, other approaches tobsAb production have included tetravalent IgG-scFv fusions (Dong e tal.,2011, MAbs 3:273-88); dual-acting Fab (DAF) antibodies (Bostrom et al.,2009, Science 323:1610-14); Igg-like dual-variable domain antibodies(DVD-Ig) (Wu et al., 2007, Nat Biotechnol 25:1290-97); and use ofdynamic exchange between IgG4 molecules (van der Neut Kolfschoten etal., 2007, Science 317:1554-57). Although the DNL® technology discussedbelow is preferred for formation of leukocyte redirecting bsAbs, theperson of ordinary skill will realize that other types of bsAbs may beused in the claimed methods and compositions.

DOCK-AND-LOCK® (DNL®)

In some embodiments, a bispecific antibody, either alone or elsecomplexed to one or more effectors such as cytokines, is formed as aDOCKANDLOCK® (DNL®) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,901,680; 7,906,118;7,981,398; 8,003,111, the Examples section of each of which isincorporated herein by reference.) Generally, the technique takesadvantage of the specific and high-affinity binding interactions thatoccur between a dimerization and docking domain (DDD) sequence of theregulatory (R) subunits of cAMP-dependent protein kinase (PKA) and ananchor domain (AD) sequence derived from any of a variety of AKAPproteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and AD peptides may beattached to any protein, peptide or other molecule. Because the DDDsequences spontaneously dimerize and bind to the AD sequence, thetechnique allows the formation of complexes between any selectedmolecules that may be attached to DDD or AD sequences.

Although the standard DNL® complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL®complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL® complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ, each of which comprises a DDD moiety amino acid sequence. The Rsubunits have been isolated only as stable dimers and the dimerizationdomain has been shown to consist of the first 44 amino-terminal residuesof RIIα (Newlon et al., Nat. Struct. Biol. 1999; 6:222). As discussedbelow, similar portions of the amino acid sequences of other regulatorysubunits are involved in dimerization and docking, each located near theN-terminal end of the regulatory subunit. Binding of cAMP to the Rsubunits leads to the release of active catalytic subunits for a broadspectrum of serine/threonine kinase activities, which are orientedtoward selected substrates through the compartmentalization of PKA viaits docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are varied amongindividual AKAPs, with the binding affinities reported for RII dimersranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA 2003;100:4445). AKAPs will only bind to dimeric R subunits. For human RIIα,the AD binds to a hydrophobic surface formed by the 23 amino-terminalresidues (Colledge and Scott, Trends Cell Biol. 1999; 6:216). Thus, thedimerization domain and AKAP binding domain of human RIIα are bothlocated within the same N-terminal 44 amino acid sequence (Newlon etal., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J. 2001;20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL®complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is stabilized with a subsequentreaction to covalently secure the two entities via disulfide bridges,which occurs very efficiently based on the principle of effective localconcentration because the initial binding interactions should bring thereactive thiol groups placed onto both the DDD and AD into proximity(Chmura et al., Proc. Natl. Acad. Sci. USA 2001; 98:8480) to ligatesite-specifically. Using various combinations of linkers, adaptormodules and precursors, a wide variety of DNL® constructs of differentstoichiometry may be produced and used (see, e.g., U.S. Pat. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL®construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Dock-and-Lock® (DNL®) technology has been used to produce a variety ofcomplexes in assorted formats (Rossi et al., 2012, Bioconjug Chem23:309-23). Bispecific hexavalent antibodies (bsHexAbs) based onveltuzumab (anti-CD20) and epratuzumab (anti-CD22) were constructed bycombining a stabilized (Fab)₂ fused to a dimerization and docking domain(DDD) with an IgG containing an anchor domain (AD) appended at theC-terminus of each heavy chain (C_(H)3-AD2-IgG) (Rossi et al., 2009,Blood 113, 6161-71). Compared to mixtures of their parental mAbs, theseFc-based bsHexAbs, referred to henceforth as “Fc-bsHexAbs”, inducedunique signaling events (Gupta et al., 2010, Blood 116:3258-67), andexhibited potent cytotoxicity in vitro. However, the Fc-bsHexAbs werecleared from circulation of mice approximately twice as fast as theparental mAbs (Rossi et al., 2009, Blood 113, 6161-71). Although theFc-bsHexAbs are highly stable ex vivo, it is possible that somedissociation occurs in vivo, for example by intracellular processing.Further, the Fc-bsHexAbs lack CDC activity.

Fc-based immunocytokines have also been assembled as DNL® complexes,comprising two or four molecules of interferon-alpha 2b (IFNα2b) fusedto the C-terminal end of the C_(H)3-AD2-IgG Fc (Rossi et al., 2009,Blood 114:3864-71; Rossi et al., 2010, Cancer Res 70:7600-09; Rossi etal., 2011, Blood 118:1877-84). The Fc-IgG-IFNα maintained high specificactivity, approaching that of recombinant IFNα, and were remarkablypotent in vitro and in vivo against non-Hodgkin lymphoma (NHL)xenografts. The T_(1/2) of the Fc-IgG-IFNα in mice was longer thanPEGylated IFNα, but half as long as the parental mAbs. Similar to theFc-bsHexAbs, the Fc-IgG-IFNα dissociated in vivo over time and exhibiteddiminished CDC, but ADCC was enhanced.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL® constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL® complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILA PKA RIIα(SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat alternative species within the genus of DDD moieties can beconstructed by standard techniques, for example using a commercialpeptide synthesizer or well known site-directed mutagenesis techniques.The effect of the amino acid substitutions on AD moiety binding may alsobe readily determined by standard binding assays, for example asdisclosed in Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50).

TABLE 2 Conservative Amino Acid Substitutions inDDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 87. S HI Q I P P G L T E L L Q G Y T V E V L R T K N A S D N A S D K R Q Q P PD L V E F A V E Y F T R L R E A R A N N E D L D S K K D L K L I I I V VV THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 12)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Other species within thegenus of possible AD moiety sequences could be made, tested and used bythe skilled artisan, based on the data of Alto et al. (2003). It isnoted that FIG. 2 of Alto (2003) shows a number of amino acidsubstitutions that may be made, while retaining binding activity to DDDmoieties, based on actual binding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 3 Conservative Amino Acid Substitutions in AD1(SEQ ID NO: 3). Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y LA K Q I V D N A I Q Q A N L D F I R N E Q N N L V T V I S VNIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA (SEQ ID NO: 37)QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA (SEQ ID NO: 41)QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA (SEQ ID NO: 43)QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA (SEQ ID NO: 45)QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV (SEQ ID NO: 49)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL® constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQA Alternative AKAPsequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA (SEQ ID NO: 52)QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 55) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 57)alyqfadrfselviseal riad (SEQ ID NO: 58) leqvanqladqiikeat pv38(SEQ ID NO: 59) feelawkiakmiwsdvf Dual-Specificity AKAPs akap7(SEQ ID NO: 60) elvrlskrlvenavlkav map2d (SEQ ID NO: 61)taeevsarivqvvtaeav dakap1 (SEQ ID NO: 62) qikqaafqlisqvileat Dakap2(SEQ ID NO: 63) lawkiakmivsdvmqq

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) wt-pep AKAP7δ-PEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) L304T-pep AKAP7δ-PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) L308D-pep AKAP7δ-PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) P-pep AKAP7δ-PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) PP-pep AKAP7δ-PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) L314E-pep AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) pep AKAP11-VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) pep AKAP12-NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) pep AKAP14-TQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) pep Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized. SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ IDNO:1)

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5 Conservative Amino Acid Substitutions in DDD1(SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 89. S H I Q IP P G L T E L L Q G Y T V E V L R T N S I L A Q Q P P D L V E F A V E YF T R L R E A R A N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL® constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Therapeutic Agents

In alternative embodiments, therapeutic agents such as cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes orother agents may be used, either conjugated to the subject bsAbs, ADCsand/or antibodies or separately administered before, simultaneouslywith, or after the bsAbs, ADCs and/or antibodies. Drugs of use maypossess a pharmaceutical property selected from the group consisting ofantimitotic, antikinase, alkylating, antimetabolite, antibiotic,alkaloid, anti-angiogenic, pro-apoptotic agents and combinationsthereof.

Exemplary drugs of use may include, but are not limited to,5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptorbinding agents, etoposide (VP16), etoposide glucuronide, etoposidephosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-ß, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -ß; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-ß; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-ß; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT.

Radionuclides of use include, but are not limited to—¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb, and ²²⁷Th. The therapeuticradionuclide preferably has a decay-energy in the range of 20 to 6,000keV, preferably in the ranges 60 to 200 keV for an Auger emitter,100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alphaemitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decayenergies of useful alpha-particle-emitting radionuclides are preferably2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably4,000-7,000 keV. Additional potential radioisotopes of use include ¹¹C,¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru,¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm,¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au,⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.Some useful diagnostic nuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or, ¹¹¹In.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Jori et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2 or p53. Apreferred form of therapeutic oligonucleotide is siRNA. The skilledartisan will realize that any siRNA or interference RNA species may beattached to an antibody or fragment thereof for delivery to a targetedtissue. Many siRNA species against a wide variety of targets are knownin the art, and any such known siRNA may be utilized in the claimedmethods and compositions.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), andapolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of eachreferenced patent incorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Minis Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject DNL® complexes.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,such as a mammal, including humans, domestic or companion pets, such asdogs and cats, comprising administering to the subject a therapeuticallyeffective amount of a combination of cytotoxic and/or immunomodulatoryagents.

The administration of the cytotoxic bsAbs, ADCs and/or checkpointinhibitor antibodies can be supplemented by administering concurrentlyor sequentially a therapeutically effective amount of another antibodythat binds to or is reactive with another antigen on the surface of thetarget cell. Preferred additional MAbs comprise at least one humanized,chimeric or human MAb selected from the group consisting of a MAbreactive with CD4, CD5, CD8, CD14, CD15, CD16, CD19, IGF-1R, CD20, CD21,CD22, CD23, CD25, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45,CD46, CD52, CD54, CD70, CD74, CD79a, CD79b, CD80, CD95, CD126, CD133,CD138, CD154, CEACAM5, CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2, carbonicanhydrase IX, PAM4 antigen, MUC1, MUC2, MUC3, MUC4, MUC5, Ia, MIF,HM1.24, HLA-DR, tenascin, Flt-3, VEGFR, PlGF, ILGF, IL-6, IL-25,tenascin, TRAIL-R1, TRAIL-R2, complement factor C5, oncogene product, ora combination thereof. Various antibodies of use, such as anti-CD19,anti-CD20, and anti-CD22 antibodies, are known to those of skill in theart. See, for example, Ghetie et al., Cancer Res. 48:2610 (1988); Hekmanet al., Cancer Immunol. Immunother. 32:364 (1991); Longo, Curr. Opin.Oncol. 8:353 (1996), U.S. Pat. Nos. 5,798,554; 6,187,287; 6,306,393;6,676,924; 7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785;7,238,786; 7,282,567; 7,300,655; 7,312,318; 7,501,498; 7,612,180;7,670,804; and U.S. Patent Application Publ. Nos. 20080131363;20070172920; 20060193865; and 20080138333, the Examples section of eachincorporated herein by reference.

The combination therapy can be further supplemented with theadministration, either concurrently or sequentially, of at least onetherapeutic agent. For example, “CVB” (1.5 g/m² cyclophosphamide,200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen usedto treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate, bendamustine, and bryostatin-1.

The combinations of therapeutic agents can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe bsAb, ADC, interferon and/or checkpoint inhibitor antibody iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON′S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

The subject bsAbs, ADCs, interferons and/or antibodies can be formulatedfor intravenous administration via, for example, bolus injection orcontinuous infusion. Preferably, the bsAb, ADC and/or antibody isinfused over a period of less than about 4 hours, and more preferably,over a period of less than about 3 hours. For example, the first boluscould be infused within 30 minutes, preferably even 15 min, and theremainder infused over the next 2-3 hrs. Formulations for injection canbe presented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic combinations. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the agents to be administered. For example, biocompatiblepolymers include matrices of poly(ethylene-co-vinyl acetate) andmatrices of a polyanhydride copolymer of a stearic acid dimer andsebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rateof release from such a matrix depends upon the molecular weight of thetherapeutic agent, the amount of agent within the matrix, and the sizeof dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The bsAbs, interferons and/or checkpoint inhibitor antibodies may beadministered to a mammal subcutaneously or even by other parenteralroutes, such as intravenously, intramuscularly, intraperitoneally orintravascularly. ADCs may be administered intravenously,intraperitoneally or intravascularly. Moreover, the administration maybe by continuous infusion or by single or multiple boluses. Preferably,the bsAb, ADC, interferon and/or checkpoint inhibitor antibody isinfused over a period of less than about 4 hours, and more preferably,over a period of less than about 3 hours.

More generally, the dosage of an administered bsAb, ADC, interferonand/or checkpoint inhibitor antibody for humans will vary depending uponsuch factors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. It may be desirable to providethe recipient with a dosage of bsAb, ADC and/or antibody that is in therange of from about 1 mg/kg to 25 mg/kg as a single intravenousinfusion, although a lower or higher dosage also may be administered ascircumstances dictate. A dosage of 1-20 mg/kg for a 70 kg patient, forexample, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosagemay be repeated as needed, for example, once per week for 4-10 weeks,once per week for 8 weeks, or once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months, ormonthly or quarterly for many months, as needed in a maintenancetherapy.

Alternatively, a bsAb, ADC, and/or checkpoint inhibitor antibody may beadministered as one dosage every 2 or 3 weeks, repeated for a total ofat least 3 dosages. Or, the combination may be administered twice perweek for 4-6 weeks. If the dosage is lowered to approximately 200-300mg/m² (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kgpatient), it may be administered once or even twice weekly for 4 to 10weeks. Alternatively, the dosage schedule may be decreased, namely every2 or 3 weeks for 2-3 months. It has been determined, however, that evenhigher doses, such as 20 mg/kg once weekly or once every 2-3 weeks canbe administered by slow i.v. infusion, for repeated dosing cycles. Thedosing schedule can optionally be repeated at other intervals and dosagemay be given through various parenteral routes, with appropriateadjustment of the dose and schedule

The person of ordinary skill will realize that while the dosageschedules discussed above are relevant for ADCs, bsAbs and/or mAbs, theinterferon agents should be administered at substantially lower dosagesto avoid systemic toxicity. Dosages of interferons (such asPEGINTERFERON) for humans are more typically in the microgram range, forexample 180 μg s.c. once per week, or 100 to 180 μg, or 135 μg, or 135μg/1.73 m², or 90 μg/1.73 m², or 250 μg s.c. every other day may be ofuse, depending on the type of interferon.

While the bsAbs, interferons, ADCs and/or checkpoint inhibitorantibodies may be administered as a periodic bolus injection, inalternative embodiments the bsAbs, ADCs, interferons and/or checkpointinhibitor antibodies may be administered by continuous infusion. Inorder to increase the Cmax and extend the PK of the therapeutic agentsin the blood, a continuous infusion may be administered for example byindwelling catheter. Such devices are known in the art, such asHICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see, e.g., Skolnik et al.,Ther Drug Monit 32:741-48, 2010) and any such known indwelling cathetermay be used. A variety of continuous infusion pumps are also known inthe art and any such known infusion pump may be used. The dosage rangefor continuous infusion may be between 0.1 and 3.0 mg/kg per day. Morepreferably, the bsAbs, ADCs, interferons and/or checkpoint inhibitorantibodies can be administered by intravenous infusions over relativelyshort periods of 2 to 5 hours, more preferably 2-3 hours.

In preferred embodiments, the combination of agents is of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor). Cancers conducive to treatment methods of thepresent invention involves cells which express, over-express, orabnormally express IGF-1R.

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational TROPhoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, TROPhoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BASICPATHOLOGY, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an antibody, antibody fragment, cytokine or constituentfusion protein of a bsAb, such as a DNL® construct. Fusion proteins maycomprise an antibody or fragment or cytokine attached to, for example,an AD or DDD moiety.

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express the encoded protein(s) in a selected host cell,enhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969). In otherembodiments, an IgG1 sequence may be converted to an IgG4 sequence.

The skilled artisan will realize that methods of genetically engineeringexpression constructs and insertion into host cells to expressengineered proteins are well known in the art and a matter of routineexperimentation. Host cells and methods of expression of clonedantibodies or fragments have been described, for example, in U.S. Pat.Nos. 7,531,327, 7,537,930, 7,785,880, 8,076,410, 8,153,433 and8,372,603, the Examples section of each incorporated herein byreference.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain one or more bsAbs, ADCs, interferons, and/or checkpointinhibitor antibodies as described herein. If the composition containingcomponents for administration is not formulated for delivery via thealimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used. In certain embodiments, atherapeutic agent may be provided in the form of a prefilled syringe orautoinjection pen containing a sterile, liquid formulation orlyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

The following examples are provided to illustrate, but not to limit, theclaims of the present invention.

Example 1 T-Cell Redirecting Bispecific Antibody DOCK-AND-LOCK® (DNL®)Complexes

Several species of exemplary leukocyte redirecting bispecific antibodieswere made as DNL® complexes, as described below. The complexes wereeffective to induce an immune response against appropriate target cellsincluding, but not limited to, Trop-2⁺ cancer cells.

Materials and Methods

General techniques for making and using DOCK-AND-LOCK® (DNL®) complexesare described in the Examples below. An exemplary leukocyte redirectingbispecific antibody with binding sites for CD3 and CD19 was made as aDNL® complex, referred to as (19)-3s (FIG. 1). An anti-CD19 F(ab)₂DNLmodule was constructed by recombinant fusion of a dimerization anddocking domain (DDD2) at the carboxyl terminal end of the Fd chain. Ananti-CD3-scFv module was designed from Okt3 mAb with addition of ananchor domain (AD2) and assembled in the formatV_(H)-L1-V_(K)-L2-6H-L3-AD2 (“6H” disclosed as SEQ ID NO:105), where theV domains were fused via a flexible peptide linker and the AD2 peptidewas preceded by a 6-His linker (SEQ ID NO:105). The sequences of theanti-CD3 variable regions, linkers and AD2 were as shown below.

V_(H )sequence of anti-CD3 scFv (SEQ ID NO: 96)QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYSLDYWGQGTTLTVSSL1 Linker (SEQ ID NO: 97) GGGGSGGGGSGGGGSV_(K )sequence of anti-CD3 scFv (SEQ ID NO: 98)DIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSG TKLEIKR L2 Linker(SEQ ID NO: 99) GGGGS Poly-His-L3 Linker (SEQ ID NO: 100) HHHHHHGGGSGAD2 (SEQ ID NO: 101) CGQIEYLAKQIVDNAIQQAGC

Expression vectors and DNL® modules—DNL® complexes were constructedcomprising antibody moieties against various disease-associatedantigens, linked to an anti-CD3 antibody moiety, generally abbreviatedas (X)-3s bsAbs. Independent production cell lines were developed inSpESFX-10 mouse myeloma cells (Rossi et al., 2011, Biotechnol Prog27:766-75) for each of the DNL® modules used to make the (X)-3s bsAbs. AcDNA sequence encoding the Okt3scFv-AD2 polypeptide (SEQ ID NOs:96-101)was synthesized and cloned into the pdHL2 expression vector via 5′ Xba Iand 3′ Eag I restriction sites. The construct comprised the V_(H) domainfused to the V_(L) in an scFv with the structureV_(H)-L1-V_(K)-L2-6H-L3-AD2 (“6H” disclosed as SEQ ID NO:105). Theexpressed protein had two amino acid substitutions from the originalOkt3 mAb. A cysteine residue in the CDR-H3 was changed to serine(Kipryanov, 1997, J Immunol Methods 200:69-77). The penultimate residueof the V_(L) was changed from aspartate to lysine.

The Okt3scFv-AD2 module was combined with various C_(H)1-DDD2-Fabmodules to generate a panel of (X)-3s trivalent bsAbs (Table 6). TheC_(H)1-DDD2-Fab-pdHL2 expression vectors were constructed as describedpreviously for similar constructs (Rossi et al., 2008, Cancer Res68:8384-92). Briefly, expression vectors encoding C_(H)1-DDD2-Fab weregenerated from the corresponding IgG-pdHL2 expression vectors byexcising the coding sequence for the C_(H)1-Hinge-C_(H)2-C_(H)3 domainswith Sac II and Eag I restriction enzymes and replacing it with a 507 bpsequence encoding C_(H)1-DDD2, which was excised from theC_(H)1-DDD2-Fab-hA20-pdHL2 expression vector (Rossi et al., 2008, CancerRes 68:8384-92) with the same enzymes. C_(H)1-DDD2-Fab modules werederived from the humanized mAbs hA19 (anti-CD19), labetuzumab (hMN-14,anti-CEACAM5), clivatuzumab (hPAM4, anti-mucin), hMN-15 (anti-CEACAM6),hRS7 (anti-TROP-2), veltuzumab (hA20, anti-CD20), hL243 (anti-HLA-DR)and epratuzumab (hLL2, anti-CD22). The mAb designated hA19 was humanizedfrom the mouse anti-CD19 mAb B43 (Uckun et al., 1988, Blood 71:13-29).Each expression vector was linearized by digestion with Sal Irestriction enzyme and used to transfect SpESFX-10 cells byelectroporation.

Clones were selected in media containing 0.2 μM methotrexate (MTX) andscreened for protein expression by ELISA. Okt3scFv-AD2 was captured onNi-NTA HisSorb plates (Qiagen) and detected with an anti-AD2 mAb.C_(H)1-DDD2-Fab modules were captured with goat-anti-human-kappa chainand detected with goat-anti-human-F(ab′)₂-HRP. Productivity ofprotein-expression was amplified by stepwise increases in MTXconcentration up to 3 μM. Okt3scFv-AD2 and C_(H)1-DDD2-Fab modules werepurified to homogeneity from the broth of roller bottle cultures byaffinity chromatography using Ni-SEPHAROSE® and Kappa-Select resins,respectively. The DNL® method was used to assemble (X)-3s bsAbs via thesite-specific conjugation of mole equivalents of Okt3scFv-AD2 andC_(H)1-DDD2-Fab modules. For example, approximately 100 mg of (19)-3swere produced by combining 22 mg of Okt3scFv-AD2 with 80 mg ofC_(H)1-DDD2-Fab-hA19. The mixture was reduced overnight at roomtemperature with 1 mM reduced glutathione prior to the addition of 2 mMoxidized glutathione. The (19)-3s was purified from the reaction mixtureby sequential affinity chromatography with Kappa-Select andNi-SEPHAROSE®. Additional (X)-3s constructs were assembled at variousscales following a similar process.

TABLE 6 (X)-3s DNL ® Constructs Code Target C_(H)1-DDD2-Fab AD2-anti-CD3(19)-3s CD19 C_(H)1-DDD2-Fab-hA19 scFv-AD2-Okt3 (20)-3s CD20C_(H)1-DDD2-Fab-hA20 scFv-AD2-Okt3 (22)-3s CD22 C_(H)1-DDD2-Fab-hLL2scFv-AD2-Okt3 (C2)-3s HLA-DR C_(H)1-DDD2-Fab-hL243 scFv-AD2-Okt3 (M1)-3sMUC5AC C_(H)1-DDD2-Fab-hPAM4 scFv-AD2-Okt3 (14)-3s CEACAM5C_(H)1-DDD2-Fab-hMN-14 scFv-AD2-Okt3 (15)-3s CEACEAM6C_(H)1-DDD2-Fab-hMN-15 scFv-AD2-Okt3 (E1)-3s TROP-2 C_(H)1-DDD2-Fab-hRS7scFv-AD2-Okt3

Analytical Methods—Size-exclusion high-performance liquid chromatography(SE-HPLC) was performed with an Alliance HPLC System with a BIOSUITE™250, 4-μm UHR SEC column (Waters Corp). Electrospray ionization time offlight (ESI-TOF) liquid chromatography/mass spectrometry (LC-MS) wasperformed with a 1200-series HPLC coupled with a 6210 TOF MS (AgilentTechnologies, Santa Clara, Calif.). The (19)-3s was resolved by reversedphase HPLC (RP-HPLC) at 60° C., using a 14-min gradient of 30-80%acetonitrile in 0.1% aqueous formic acid with an Aeris widepore 3.6 μmC4 column (Phenomenex). For the TOF MS, the capillary and fragmentorvoltages were set to 5500 and 300 V, respectively.

Cell Lines and Reagents—Raji, Ramos, Daudi, LS174T and Capan-1 celllines were purchased from the American Type Cell Culture Collection(ATCC, Manassas, Md.) and Nalm-6 cells were purchased from DeutscheSammlung von Mikroorganismen and Zellinien (DSMZ, Braunchweig, Germany).All cell lines, except Capan-1, were maintained in RPMI-1640 containing10% FBS, 1% L-glutamine, 1% penicillin-streptomycin and 1% MEMnonessential amino acids. Capan-1 cells were maintained with 20% FBS.All cell culture media and supplements were purchased from LifeTechnologies (Carlsbad, Calif.).

PBMCs and T cell isolation—Human peripheral blood mononuclear cells(PBMC) were purified from whole donor blood (Blood Center of NJ, EastOrange, N.J.) using UNI-SEP_(MAXI) tubes (Novamed, Ltd, Jerusalem,Israel). CD3-positive T cells were isolated from PBMCs by negativeselection using the Pan T Cell Isolation Kit (Miltenyi Biotec, Auburn,Calif.), according to the manufacturer's protocol. Efficiency of T cellisolation was assessed by FACS after staining the enriched T cells withanti-CD3-PE antibody. In some cases, further staining with CD-19 andCD-14 was performed as well to identify contaminating cells.

T cell activation—Isolated T cells were plated in 6-well tissue cultureplates at a final density of 2.25×10⁶ cells/well. Daudi cells were addedto some wells at a final density of 1.5×10⁶ cells/well, other wells wereleft to contain only T cells. Alternatively, PBMCs were added to 6-welltissue culture plates at a final cell density of 6×10⁶ cells/well. Thevolume of each well was brought up to 3 mL. To the appropriate wells, 3ng/mL of (19)-3s, (M1)-3s or (19)-DDD2 was added. After incubationovernight at 37° C., 1 mL of each sample was removed. The cells werepelleted and labeled on ice with CD69-APC and CD3-PE for 20 minutes.Cells were washed 2 times with 1% BSA in PBS and analyzed using aFACSCALIBER™ flow cytometer (BD Biosciences, San Jose, Calif.).

T-cell proliferation—PBMCs were seeded in T25 flasks at a concentrationof 1×10⁶ cells/mL containing the specified reagents. For B cell-depletedflasks, B cells were removed by negative selection using a B-cellisolation kit from Miltenyi according to manufacturer's protocol. Onselect days, 100 μL of media was removed from each flask, labeled withanti-CD7-APC for 20 minutes on ice, washed once and resuspended in 300μL of 1% BSA/PBS containing 7-AAD. For each sample, the entire volume isanalyzed using a FACSCALIBER™ flow cytometer. Each sample is counted induplicate. Analysis is performed using FlowJo Software. For each sample,dead (7-AAD+) cells, and debris (based on forward vs. side scatter) wasremoved. Finally, live CD7+ cells were selected and plotted using Prismsoftware.

Cell Binding Assays (Jurkat/Capan-1)—Jurkat cells were stained withPKH26 Red Fluorescent Cell Linker Kit (Sigma) according tomanufacturer's protocol. Capan-1 cells were stained with 5 μM CFSE(carboxyfluorescein diacetate succinimidyl ester, Life Technologies)according to manufacturer's protocol. Labeled Capan-1 cells were addedto 8-well chamber slides (Thermo Waltham, Mass.) and allowed to attachovernight. The following day, media was removed and PKH26-labeled Jurkatcells were added in media containing 0.1 μg/mL of (E1)-3s, (M1)-3s or(19)-3s. Following a 1-hour incubation at 37° C., slides were washedwith PBS to remove any unbound cells and observed by fluorescencemicroscopy.

Cell Binding Assays (Jurkat/Daudi)—Jurkat and Daudi cells were labeledwith anti-CD3-PE and anti-CD20-FITC, respectively. Labeled cells werethen coincubated at a 2.5:1 ratio with 0.1 μg/mL (19)-3s for 30 minutesat room temperature. Aliquots of cells were then observed byfluorescence microscopy.

Cytotoxicity Assay (Hematologic Tumor Cell Lines)—Target cells werelabeled with PKH67 Green Fluorescent Cell Linker Kit (Sigma) accordingto the manufacturer's protocol. Briefly, 5×10⁶ target cells wereresuspended in 250 μL of diluent C. In a second tube 1 μL of PKH26 dyeis added to 250 μL of diluent C. The cell suspension is then added tothe dye solution, mixed thoroughly and incubated at RT for 2 minutes.The reaction was quenched by adding an equal volume of FBS. The labeledcells were then washed 3 times with complete RPMI. Unstimulated,isolated T cells were used as effector cells. Effector cells andPKH67-labeled target cells were combined at a 10:1 ratio and plated in48-well plates containing serial dilutions of (19)-3s or (14)-3s. Eachwell contained 5×10⁴ target cells and 5×10⁵ effector cells. Jeko-1assays were performed in 20% RPMI. Plates were incubated for 18-24 hoursin a 37° C. incubator containing 5% CO₂. Following incubation, all cellswere removed from 48-well plates into flow cytometer tubes andresuspended in 1% BSA/PBS containing 1 ug/mL of 7AAD, to distinguishlive from dead cells, and 30,000 COUNTBRIGHT™ Absolute Counting Beads(Life Technologies). Cells were analyzed on a FACSCALIBER™ flowcytometer. For each sample, 8,000 COUNTBRIGHT™ beads were counted as anormalized reference. Data were analyzed using FlowJo software(Treestar, Inc., Ashland, Oreg.). For each sample, dead cells and debriswere excluded and total live target cells were counted.

Cytotoxicity Assay (Solid Tumor Cell Lines)—Target cells were labeledwith PKH67 Green Fluorescent Cell Linker Kit (Sigma) following the sameprocedure as for staining with PKH23. Effector cells used were asfollows: For Capan-1 assays, CD8+ enriched T cells were used, followingpurification from a CD8+ enrichment column (R&D Systems, Minneapolis,Minn.). For LS174T cells: Stimulated T cells were used after incubationof PBMC for 5 days in media containing 25 U/mL IL-2 and 50 ng/mL Okt3Mab, followed by 2 days incubation in media containing 25 U/mL IL-2alone. Effector cells and PKH67-labeled target cells were combined at a3:1 ratio (5×10⁴ target cells and 1.5×10⁵ effector cells/well) andplated over 48-well plates containing serial dilutions of (E1)-3s,(14)-3s or (19)-3s. Capan-1 assays were performed in 20% RPMI. Plateswere incubated for 42-48 hours in a 37° C. incubator containing 5% CO₂.Following incubation, suspension cells were combined with trypsinizedattached cells from all wells and transferred into flow cytometer tubes.Cells were washed one time and resuspended in 1% BSA/PBS containing 1ug/mL of 7AAD, to distinguish live from dead cells, and 30,000COUNTBRIGHT™ Absolute Counting Beads. Cells were analyzed on aFACSCALIBER™ flow cytometer. For each sample, 8,000 COUNTBRIGHT™ beadswere counted as a normalized reference. Data were analyzed using FlowJosoftware (Treestar, Inc., Ashland, Oreg.). For each sample, dead cellsand debris were excluded and total live target cells were counted.

In Vivo Efficacy—Female NOD/SCID mice, 8 weeks old, were purchased fromCharles River (Wilmington, Mass.). Mice were injected s.c. with amixture of Raji (1×10⁶) and human PBMCs (5×10⁶ cells) mixed 1:1 withmatrigel. Therapy began 1 hour later. Treatment regimens, dosages, andnumber of animals in each experiment are described in the Results.Animals were monitored daily for signs of tumor out-growth. Once tumorsappeared, they were measured twice weekly. Tumor volume (TV) wasdetermined by measurements in two dimensions using calipers, withvolumes defined as: L×w²/2, where L is the longest dimension of thetumor and w the shortest. Efficacy was determined by a log-rank testusing Prism GraphPad software (v5; LaJolla, Calif.) on Kaplan-Meiercurves using survival surrogate endpoints as time for tumor progression(TTP) to 1.0 cm³. Significance was considered at P<0.05.

Results

Construction and biochemical analysis of leukocyte redirectingbispecific antibodies. The DNL® method was used to generate a panel of(X)-3s, leukocyte redirecting bsAbs for targeting of varioustumor-associated antigens including CD19, CD20, HLA-DR, TROP-2, CEACAM5and MUC5AC. The purity of these structures was demonstrated by SE-HPLCand SDS-PAGE analysis, where only bands representing the threeconstituent polypeptides (Okt3scFv-AD2, hA19-Fd-DDD2 and hA19 kappa)were evident (data not shown). LC-MS analysis identified a singleRP-HPLC peak having a deconvoluted mass spectrum consistent (massaccuracy=11 ppm) with the calculated mass (137432.37 Da) of (19)-3s fromits deduced amino acid sequence, including the predicted amino-terminalpyroglutamates on the Okt3scFv-AD2 and each of the two C_(H)1-DDD2-hA19Fd chains (data not shown). No additional post-translationalmodifications, including glycosylation were indicated.

Immune synapse formation between Daudi Burkitt lymphoma and T cells,mediated by (19)-3s. The effects of the leukocyte redirecting (19)-3sDNL® complex on targeting effector T cells to CD19⁺ lymphoma cells wasexamined (FIG. 2). Freshly isolated T cells were combined with Daudicells at an E:T ratio of 2.5:1. Cells were treated with 0, 1 or 5 μg/mLof (19)-3s DNL® complex for 30 min at room temperature prior to analysisby flow cytometry. Anti-CD20-FITC and anti-CD7-APC were used to identifyDaudi and T cells, respectively. Co-binding was indicated as the % ofCD20⁺/CD7⁺ events. After treatment with (19)-3s, 45.5% of flow eventswere CD20/CD7 dual-positive, indicating synapsed Daudi and T cells (FIG.2A), compared to 2% measured for the mixed cells without antibody (FIG.2B). Addition of (19)-3s resulted in association of >90% of the Daudiwith T cells (FIG. 2C). These results show that the (19)-3s DNL® complexwas effective to direct T cells to the targeted antigen-expressinglymphoma cells.

Synapse formation between T cells and target lymphoma cells wasdemonstrated by fluorescence microscopy (FIG. 3) Jurkat (T cells) andDaudi (B cells) were combined at a 1:1 ratio, treated with 0.1 μg/mL ofthe (19)-3s DNL® complex for 30 minutes and stained with anti-CD20-FITC(FIG. 3A) and anti-CD3-PE (FIG. 3B), prior to analysis by fluorescencemicroscopy. The merged image (FIG. 3C) reveals synapse formation betweengreen-stained Daudi and red-stained Jurkat cells. Synapse formation wasnot evident in the absence of (19)-3s (FIG. 3D). FIG. 3C demonstratesthat the target lymphoma cells are in direct contact with the targeted Tcells.

A dose-response series was performed for (19)-3s mediated association ofT cells to an exemplary B-cell lymphoma line (FIG. 4). As shown in FIG.4, under the conditions of this experiment, saturation of(19)-3s-mediated cell-to-cell association of T cells to target cells wasreached at a concentration between 0.037 and 0.111 μg/ml of the DNL®complex.

FIG. 5 shows a comparision of the relative efficacies of BITE® (FIG.5A), DART™ (FIG. 5A) and DNL® (FIG. 5B) anti-CD3×anti-CD19 complexes forredirecting T cells to targeted CD19⁺ B cells. The data for BITE® andDART™ was obtained from Moore et al. (2011, Blood 117:4542-51). At thelowest concentration tested of 0.0005 μg/ml, the (19)-3s DNL® complexwas more effective than BITE® or DART™ at targeting T cells to B-celllymphoma (FIG. 5). The (19)-3s DNL® complex also induced a slightlyhigher maximum level of cell-to-cell association than the comparableBITE® and DART™ complexes (FIG. 5A). Although difficult to extrapolatefrom the single data points generated for the (19)-3s DNL® complex, theEC₅₀ levels appeared to be similar for BITE®, DART™ and DNL® (FIG. 5).

(19)-3s, (E1)-3s and (M1)-3s-mediated cell-cell association of T cellsto target tumor cells. To evaluate the ability of the T-cell redirectingBsAbs to facilitate the association of T cells to their target tumorcells, Jurkat T cells were coincubated with target tumor cellscontaining (X)-3s and evaluated by flow cytometry and fluorescencemicroscopy. Jurkat T cells are a CD4+ T cell leukemia line, chosen fortheir ability to demonstrate T cell binding without depletion of theFITC labeled Daudi cells in the presence of various concentrations of(19)-3s and analyzed by flow cytometry for the detection of doublepositive (CD3+CD20+) populations indicating T cell-B cell associatedcomplexes. An apparent cell-cell association was seen followingtreatment with 0.5 ng/mL of (19)-3s and after treatment with 0.1 μg/mLover 25% of the cell population existed in a cell-cell association (FIG.5). Fluorescent microscopy supports this data, as immune synapses areevident following treatment with 0.1 μg/mL (19)-3s (FIG. 4). No synapseformation was seen in the absence of (19)-3s (data not shown).

This cell-cell association was observed in the pancreatic tumor lineCapan-1 as well (FIG. 6). Capan-1 expresses high levels of TROP2 andmoderate levels of MUC5AC. Therefore, both the TROP2-targeting bsAb,(E1)-3s (FIG. 6C), and MUC5AC-targeting bsAb, (M1)-3s (FIG. 6B) werecompared to the non-targeting control bsAb, (19)-3s (FIG. 6A).CFSE-labeled Capan-1 cells were coincubated with PKH26-labeled Jurkat inthe presence of these bsAbs. Fluorescent microscopy revealed, asexpected, large T-cell/Capan complexes mediated by (E1)-3s, followed bysmaller, yet substantial complexes mediated by (M1)-3s and relativelylow complex formation following (19)-3s treatment (FIG. 6).

(19)-3s specifically induces T cell activation and proliferation. Theability of (19)-3s to activate T cells was evaluated either in PBMCs(FIG. 7A), or T cells coincubated with Daudi B cells (FIG. 7B), bymeasuring the expression levels of CD69, an early marker of T cellactivation. Treatment with 3 ng/mL of (19)-3s induced T cell activationin T cells coincubated with Daudi B cells as indicated by a >50-foldincrease in CD69 expression compared with non-targeting controlantibodies, (19)-DDD2 and (M1)-3s, as well as T cells treated with(19)-3s without Daudi target cells (FIG. 7B). Similar results wereobserved when the antibodies were incubated with PBMCs, containing bothT and B cells; (19)-3s stimulated CD69 expression levels >20-fold higherthan non-targeting controls (FIG. 7A). In the absence of target cells,purified T cells treated with (19)-3s did not show activation (FIG. 7C).

T cell proliferation, as another indication of T cell activation, wasevaluated after treatment of PBMCs with various CD3-targetingantibodies. (19)-3s at 3 nM or 30 pM induced T cell proliferationsimilar to that of the positive control IL-2/PHA (FIG. 8A).Non-targeting control antibody, (14)-3s, shows some non-specific T cellproliferation at the highest (3 nM) concentration (FIG. 8A). However, Tcell proliferation was not observed in PBMCs depleted of B cells (FIG.8B), suggesting that target cells are necessary for specific (19)-3sinduced T cell proliferation.

(X)-3s re-directed T-cell mediated killing of malignant cell lines. Thecytotoxicity of each leukocyte targeting molecule was evaluated by itsability to mediate lysis of specific tumor target cells. For thehematologic tumor cell lines, a 10:1 E:T ratio using an unstimulated,enriched T cell population as the effector cells in an 18-24 hour assaydemonstrated the optimal assay conditions. The CD19-targeting bsAb,(19)-3s induced the most potent specific killing of the relatively lowCD19-expressing cell lines Ramos (IC₅₀=0.17 pM, Lysis_(Max)=79%) Daudi(IC₅₀=1 pM, Lysis_(Max)=60%), and Nalm6 (IC₅₀=6 pM, Lysis_(Max)=93%)(FIG. 9A). Interestingly, the high CD19-expressing cell lines, Namalwa(IC₅₀=63 pM, Lysis_(Max)=60%) and Raji (IC₅₀=3 nM, Lysis_(Max)=41%) werethe least sensitive to (19)-3s (FIG. 9A). The non-targeting (14)-3s DNL®construct had little cytotoxic effect in any of the cell lines tested(FIG. 9B). Consistent cytotoxic effects of the (19)-3s construct on theNalm-6 ALL cell line were obtained with PBMCs obtained from twodifferent donors (FIG. 9C).

The in vitro cytotoxic effects of (20)-3s, (22)-3s and (C2)-3s T-cellredirecting bsAbs were determined in several cell lines (FIG. 10). TheCD22-targeting bsAb, (22)-3s, demonstrated potent (IC₅₀=5 pM,Lysis_(Max)=60%) specific T-cell mediated lysis in the CD22-positiveDaudi cell line (FIG. 10C), but not in the CD22-negative Namalwa cells(FIG. 10A).

The CD20-targeting bsAb, (20)-3s demonstrated the highest potency in thehigher-expressing CD20 cell lines, Daudi (IC₅₀=<0.3 pM, Lysis_(Max)=90%)(FIG. 10C) and Jeko (IC₅₀=1 pM, Lysis_(Max)=90%) (FIG. 10B), compared tothe lower CD20-expressing Namalwa cell line (IC₅₀=30 pM,Lysis_(Max)=53%) (FIG. 10A).

The HLA-DR-targeting bsAb, (C2)-3s was tested in the HLA-DR expressingJeko-1 cell line (IC₅₀=20 pM, Lysis_(Max)=88%) (FIG. 10B).

At an E:T ratio of 10:1, using isolated T cells as effector cells, thebsAbs induced potent T cell-mediated cytotoxicity in various B cellmalignancies, including Burkitt lymphoma (Daudi, Ramos, Namalwa) mantlecell lymphoma (Jeko-1) and acute lymphoblastic leukemia (Nalm-6) (Table7). A non-tumor binding control, (14)-3s, induced only moderate T-cellkilling at >10 nM. The nature of the antigen/epitope, particularly itssize and proximity to the cell surface, appears to be more importantthan antigen density for T-cell retargeting potency (Table 7). It islikely that (20)-3s is consistently more potent than (19)-3s and(C2)-3s, even when the expression of CD19 or HLA-DR is considerablyhigher than CD20, as seen with Namalwa and Jeko-1, respectively (Table7). This is likely because the CD20 epitope comprises a smallextracellular loop having close proximity to the cell surface. Whencompared directly using Daudi, (22)-3s was the least potent. Compared toCD19 and CD20, CD22 is expressed at the lowest density, is a rapidlyinternalizing antigen, and its epitope is further away from the cellsurface. Each of these factors may contribute to its reduced potency.Finally, sensitivity to T-cell retargeted killing iscell-line-dependent, as observed using (19)-3s, where Raji (IC₅₀>3 nM)is largely unresponsive yet Ramos (IC₅₀=2 pM) is highly sensitive, eventhough the former expresses higher CD19 antigen density (Table 7).

In conclusion, (19)-3s, (20)-3s, (22)-3s and (C2)-3s bind to T cells andtarget B cells simultaneously and induce T-cell-mediated killing invitro. The modular nature of the DNL method allowed the rapid productionof several related conjugates for redirected leukocyte killing ofvarious B cell malignancies, without the need for additional recombinantengineering and protein production. The close proximity of the CD20extracellular epitope to the cell surface resulted in the highestpotency for (20)-3s.

TABLE 7 Ex vivo re-directed T-cell killing Antigen Expression² IC₅₀ ⁴(pM) Cell Line Type¹ CD19 CD20 CD22 HLA- (19)-3s (20)- (22)-3s (C2)-Daudi BL 1.00 1.00 1.00 1.00 1 0.3 6 N.D. Ramos BL 0.76 0.65 0.26 0.36 20.4 N.D.  2 Nalm-6 ALL 1.63 0.05 0.19 0.17 6 N.D. N.D. N.D. Namalwa BL0.76 0.11 0.05 0.40 63 30 >3000 N.D. Raji BL 1.41 0.69 0.59 0.84 >3000N.D. N.D. N.D. Jeko-1 MCL 0.89 1.02 0.05 1.06 3000 1 N.D. 20 ¹BL,Burkitt lymphoma; ALL, acute lymphoblastic leukemia; MCL, mantle celllymphoma. ²Expression level determined by flow cytometry and normalizedto that of Daudi. ³IC₅₀, the picomolar concentration that achieved 50%target cell killing.

The in vitro cytotoxic effects of leukocyte redirecting bsAbs were alsodetermined in solid tumor cells (FIG. 11). For the solid tumor celllines, optimal assay conditions were determined to be a 3:1 E:T ratiousing stimulated T cells in a 42-48 hour assay. Each bsAb inducedspecific T-cell mediated lysis of the tumor target cells. TheCEACAM5-expressing human colon adenocarcinoma cell line, LS-174T,demonstrated potent specific lysis (IC₅₀=2 pM) following treatment with(14)-3s (FIG. 11A). (E1)-3s mediated potent specific lysis of the TROP2expressing Capan-1 human pancreatic adenocarcinoma cell line (IC₅₀=29pM) (FIG. 11B). The gastric carcinoma cell line NCI-N87, which expresseshigh levels of both CEACAM6 and TROP 2 demonstrated very potent specificlysis to both T-cell targeting molecules, (15)-3s and (E1)-3s (IC₅₀=3 pMand 0.85 pM respectively) (FIG. 11C). The non-targeting controlantibody, (19)-3s, induced low (<20%) non-specific lysis atconcentrations >1 nM for Capan-1 and LS174T, and moderate (˜40%)non-specific lysis in NCI-N87 cells (FIG. 11A-C). A summary of the invitro cytotoxicity data for various leukocyte redirecting bsAbs in avariety of tumor cell lines is shown in FIG. 12. The various constructsshowed a maximal cell lysis of up to 90% or more of the targeted tumorcells, with IC₅₀ values for cell lines expressing the targeted antibenthat were generally in the low picomolar range (FIG. 12).

Example 2 In Vivo Studies of Leukocyte Redirecting DNL® Complex

One potential limitation of small (<60 kDa) scFv-based constructs, suchas BITE® and DART™, is the requirement for administration by long-termcontinuous infusion, due to their toxicity and rapid clearance fromcirculation. Because the molecular size of DNL® bsAbs is above thethreshold typically associated with renal clearance, it should exhibitslower clearance from circulation. We measured the pharmacokineticparameters in mice following a single bolus i.v. injection of 5 mg/kg ofthe (19)-3s bsAb (data not shown). A biphasic clearance was observedwith a t½α and t½β of 1.1 and 5.1 h, respectively, resulting in an areaunder the curve of 1880 pmol*h/mL (data not shown), which was nearly6-fold greater than that reported for MT103 (anti-CD19×anti-CD3 BITE®)administered at the same molar concentration (US PatentUS2010/0303827A1). The major difference is apparently a longer t½α for(19)-3s (data not shown). Because of the potentially advantageousproperties of (19)-3s, we evaluated the possibility of using lessfrequent dosing schedules rather than daily dosing, which is typicallyused for BITE® in animal studies.

A pilot study was performed using Raji human Burkitt lymphoma xenograftsin NOD/SCID mice reconstituted with human PBMCs (FIG. 13, FIG. 14). Rajicells (1×10⁶ cells/mouse) were combined with freshly isolated PBMCs(5×10⁶ cells/mouse) from a single healthy donor, mixed 1:1 withmatrigel, and injected SC into all of the animals in the study on Day 0.Groups of 5 mice received i.v. injections of (19)-3s totaling 130 μg asa single dose on Day 0 (FIG. 13B), three doses of 43 μg (Days 0, 2 and4) (FIG. 13C) or five daily doses of 26 μg (Days 0-5) (FIG. 13D). Theuntreated group (FIG. 13A), which was inoculated with the same cellmixture but did not receive (19)-3s, had a median survival time (MST) of31 days. Each therapy regimen improved survival (P≤0.05), with the threedose (every other day) schedule providing the greatest survival benefit(MST=91 days; P=0.0018 by log-rank analysis).

A follow-up study was begun to determine the efficacy of less frequentdosing (FIG. 14). Groups of 9 NOD/SCID mice were inoculated with Rajiand PBMCs in a similar fashion as above. In this study, therapy wasextended to two weeks, compared to one week in the first study. Groupsreceived i.v. injections of (19)-3s totaling 360 μg as 2×130-μg (FIG.14B), 4×65-μg (FIG. 14D) or 6×43-μg doses over two weeks (FIG. 14E). Anadditional group was administered 2×130-μg doses SC, instead of i.v.(FIG. 14C). For comparison, control groups of untreated mice (FIG. 14A)or mice treated with non-targeting (M1)-3s antibody (FIG. 14F) wereprepared. As of Day 28, each of the (19)-3s treatment groups hadsignificantly smaller AUC than the untreated control (P<0.05).Surprisingly, two weekly doses via the SC route was apparently aseffective as greater frequency i.v. dosing.

In vivo studies were also performed using solid tumors (FIG. 15).NOD/SCID mouse xenografts were prepared as described above, for theLS174T colon adenocarcinoma (FIG. 15A, FIG. 15B) or Capan-1 pancreaticcarcinoma (FIG. 15C, FIG. 15D). In each case, mice administered thetargeting (E1)-3s (FIG. 15B) or (14)-3s (FIG. 15D) bsAb DNL® constructsshowed improved survival compared to controls.

In conclusion, the leukocyte-retargeting bsAbs, including (19)-3s,(E1)-3s and (M1)-3s DNL® constructs, mediated synapse formation betweenT cells and B cells, colon adenocarcinoma or pancreatic carcinoma cells,respectively, via monovalent and bivalent binding to CD3 and CD19,respectively. T-cell activation, proliferation and target cell killingwere induced by the DNL® bsAbs at pM concentrations in an ex vivosetting. Advantageous properties of the DNL® bsAbs, including bivalenttumor binding and slower clearance, would allow for less frequent dosingand possibly SC administration, compared to BITE® or DART™ constructs,which are administered i.v. daily in animal models and as a continuousinfusion in the clinic. The modular nature of the DNL® method allows therapid production of a large number of related conjugates for redirectedleukocyte killing of various malignancies, without the need foradditional recombinant engineering and protein production.

The person of ordinary skill in the art will realize that otherantibodies that bind to CD3 or other leukocyte antigens, as well asother antibodies that bind to Trop-2 or other disease-associatedantigens are known in the art and any such antibody can be used to makeF(ab)₂, scFv or other antibody fragments using techniques well known inthe art. Such alternative antibodies or fragments thereof may beutilized in the instant methods and compositions. As discussed below,methods of making DOCK-AND-LOCK™ (DNL®) complexes may be applied toincorporate any known antibodies or antibody fragments into a stable,physiologically active complex.

Example 3 Interferon-α Enhances the Cytotoxic Effect ofAnti-Trop-2×Anti-CD3 Bispecific Antibodies

The therapeutic efficacy of an anti-human Trop-2×anti-human CD3bispecific antibody ((E1)-35), made from hRS7 and OKT3 as a DNL®complex, was tested for its ability to delay tumor outgrowth of Capan-1human pancreatic adenocarcinoma tumor cells when mixed with humanT-cells and injected into mice. The effect of interferon-α (either inthe form of E1*-2b or PEGASYS®) when combined with this therapy was alsoevaluated.

Methods

Five week-old female NOD/SCID mice were injected s.c. with a mixture ofCapan-1 (5×10⁶) and human T-cells (2.5×10⁶ cells) mixed 1:1 withmatrigel (E:T ratio of 1:2). There were six different treatment groupsof 8 mice each. Treatment consisted of one group receiving 47 μg (E1)-3si.v. every day for five days starting 1 hour after the administration ofthe Capan-1/T-cell mixture. Two groups were treated with equimolaramounts of IFN, one received the DNL molecule made fromIFN-α2b-DDD2-CK-hRS7 IgG1 (E1*-2b; 2.5 μg s.c. weekly×4 wks) whileanother received PEGASYS® (Roche; 0.6 μg s.c. weekly×4 wks). Two othergroups received a combination of (E1)-3s plus E1*2b or (E1)-3s plusPEGASYS®. The final group control group remained untreated. Table 8summarizes the various treatment groups.

TABLE 8 Treatment Groups for (E1)-3s Therapy (E1)-3s Therapy of a HumanPancreatic Carcinoma Xenograft (Capan-1) in NOD/SCID Mice Group (N)Amount Injected Schedule 1 8 Untreated N.A. 2 8 (E1)-3s qdx5 (47 μgi.v.) 3 8 E1*-2b qwkx4 (2.5 μg s.c.) 4 8 PEGASYS ® qwkx4 (0.6 μg s.c.) 58 (E1)-3s + qdx5 + E1*-2b qwkx4 6 8 (E1)-3s + qdx5 + PEGASYS qwkx4

Mice were monitored daily for signs of tumor out-growth. All animals hadtheir tumors measured twice weekly once tumors began to come up. Micewere euthanized for disease progression if their tumor volumes exceeded1.0 cm³ in size.

Results

Mean tumor volumes for the various groups are shown in FIG. 16. The datacontaining PEGASYS® groups (FIG. 16B) are shown on a separate graph fromthe E*2b groups (FIG. 16A) for clarity. All treatments weresignificantly better at controlling tumor growth in terms ofarea-under-the-curve (AUC) when compared to the untreated mice out today 29, which was when the first mouse in the untreated group waseuthanized for disease progression (P<0.0009; AUC_(29 days)). Combining(E1)-3s with PEGASYS® resulted in the best anti-tumor response overallin terms of tumor out-growth (FIG. 16B). This treatment wassignificantly better than any of the individual treatments (P<0.042;AUC) as well as superior to the combination of (E1)-3s plus E1*-2b(P=0.0312; AUC_(53 days)) (FIG. 16A). The combination of (E1)-3s plusE1*2b could significantly control tumor growth when compared to E1*2b orPEGASYS® alone (P<0.0073; AUC_(46 days)) but not (E1)-3s alone (FIG.16A-B). There were no significant differences between mice treated with(E1)-3s, PEGASYS®, or E1*-2b (FIG. 16A-B).

In terms of survival, all treatments provide a significant survivalbenefit when compared to the untreated mice (P<0.0112; log-rank) (FIG.17). As of day 81, there was no significant difference in mediansurvival times (MST) between mice treated with the combination of(E1)-3s plus E1*-2b and those treated (E1)-3s plus PEGASYS® (MST=79.5and >81 days, respectively) (FIG. 17). The mice treated with (E1)-3splus PEGASYS® had a significantly improved survival outcome than any ofthe individual treatments (P<0.0237) (FIG. 17). Mice treated with(E1)-3s plus E1*2b had a survival benefit when compared to mice treatedwith E1*-2b alone (MST=53 days; P<0.0311) but not when compared to micetreated with just (E1)-3s or PEGASYS® alone (MST=68 and 53 days,respectively) (FIG. 17). Treatment with (E1)-3s provided a significantimprovement in survival when compared to mice treated with E1*-2b(P=0.0406) but not when compared to mice treated with PEGASYS® alone(FIG. 17). There was no significant differences between mice treatedwith only E1*2b and those treated with PEGASYS® alone (FIG. 17).

The results demonstrate that addition of interferon-α provides asubstantial increase in survival and decrease in tumor growth whencombined with a leukocyte redirecting bsAb. The person of ordinary skillwill realize that the improved efficacy observed with addition of type Ior type III interferons (interferon-α, interferon-β, or interferon-λ) isnot limited to the specific (E1)-3s bsAb, but will be observed withother leukocyte redirecting bsAbs, made either as DNL® complexes or inother forms, such as BITE® or DART™.

Example 4 Further Studies on Interferon-α Combination Therapy withLeukocyte-Redirecting Bispecific Antibodies

In the Example above, the combination of (E1)-3s plus PEGASYS® proved tobe a very effective treatment in the control of tumor growth. In orderto confirm these results and extend them, a study was performed in whichtwo new groups were added. First, a control group for (E1)-3s wasincluded in which an equimolar amount of TF12 was administered toanimals. TF12 consists of two hRS7-Fab molecules linked to onenon-targeting 679 Fab (anti-HSG). Additionally, since Capan-1 issensitive to IFN, another group was added in which the effect ofPEGASYS® on Capan-1 tumor growth was assessed without the benefit of Tcells. After the mice (40) were injected with the Capan-1/T-cellmixture, they were randomized into five treatment groups. One hourlater, one group of 11 mice received 47 μg (E1)-3s i.v. every daystarting 1 h post-tumor cell injection and continued for four moreconsecutive days (qd×5). One group of 7 animals received interferon inthe form of PEGASYS® s.c. on a weekly basis for four weeks. Anothergroup received a combination of (E1)-3s i.v. plus PEGASYS® s.c.Untreated control animals receive Capan-1/T cells but no treatment. Afurther control group received TF12 at amounts equivalent to the (E1)-3sin terms of moles (57 μg qd×5). Group 6 mice (8 animals) received aseparate injection of only Capan-1 cells (i.e., no T cells) and wastreated with PEGASYS®. All therapy injections were in a volume of 100μL. Table 9 summarizes the various groups

TABLE 9 Treatment Groups for (E1)-3s and TF12 Therapy (E1)-3s Therapy ofa Human Pancreatic Carcinoma Xenograft (Capan-1) in NOD/SCID Mice Group(N) Amount Injected Schedule 1 7 Untreated N.A. (Capan-1 + T cells only)2 11 (E1)-3s qdx5 (47 μg i.v.) 3 7 TF12 qdx5 (57 μg i.v.) 4 7 PEGASYS ®qwkx4 (0.6 μg s.c.) 5 8 (E1)-3s + qdx5 + PEGASYS ® qwkx4 6 8 PEGASYS ®qwkx4 (0.6 μg s.c.) (Capan-1 cells only)

Mice were monitored daily for signs of tumor out-growth. All animals hadtheir tumors measured twice weekly once tumors began to come up. Micewere euthanized for disease progression if their tumor volumes exceeded1.0 cm³ in size.

Results

Mean tumor growth (FIG. 18) and survival curves (FIG. 19) are shown.While not different from each other, mice treated with (E1)-3s,PEGASYS®, or PEGASYS® (without T cells), demonstrated significantanti-tumor effects when compared to TF12 and untreated control groups(P<0.0102; AUC). On the day this experiment ended (day 59), the meantumor volume for the mice treated with the combination of (E1)-3s plusPEGASYS® was 0.083±0.048 cm³. Overall, this treatment group demonstrateda significant anti-tumor effect when compared to all the other treatmentgroups (P<0.0072; AUC).

Each individual treatment (PEGASYS®, PEGASYS® without T cells, and(E1)-3s) significantly improved survival in comparison to both the TF12and untreated control groups (P<0.0059; log-rank) (FIG. 18, FIG. 19).All the groups except the combination of (E1)-3s plus PEGASYS® reachedtheir respective MSTs. No animals were euthanized for diseaseprogression (TV>1.0 cm³) in this combination group. Importantly, thecombination of (E1)-3s plus PEGASYS® provided a significant survivalbenefit when compared to all other treatments (P<0.0007; log-rank) (FIG.18, FIG. 19).

Example 5 Effect of Interferon-α Combination Therapy withT-Cell-Redirecting Bispecific Antibodies in Human Gastric Cancer

The methods and compositions disclosed in the preceding two Exampleswere used to study the effects of leukocyte redirecting bsAbs alone orin combination with interferon-α (PEGASYS®) in the IFN-refractoryNCI-N87 human gastric tumor line. Groups of mice (N=8 each group) wereinjected s.c. with 5×10⁶ NCI-N87 cells+2.5×10⁶ T Cells (1:2 E:T ratio)mixed with matrigel and therapy started 1 h later. The treatment groupsare shown in Table 10.

TABLE 10 Treatment Groups for (E1)-3s and TF12 Therapy (E1)-3s Therapyof a Human Gastric Carcinoma Xenograft (NCI-N87) in NOD-SCID Mice GroupAmount Injected Schedule 1 Untreated (NCI-N87 + T cells only) N.A. 2(E1)-3s (47 μg i.v.) qdx5 3 TF12 (57 μg i.v.) qdx5 4 PEGASYS ® (0.6 μgs.c.) qwkx4 5 TF12 + PEGASYS ® qdx5 + qwkx4 6 (E1)-3s + PEGASYS ® qdx5 +qwkx4

The effects of leukocyte redirecting bsAb (E1)-3s alone or incombination with interferon are shown in FIG. 20 and FIG. 21. The(E1)-3s bsAb was effective to reduce tumor growth and increase survivalin gastric cancer. Significantly, the combination with interferon-αenhanced the effect of leukocyte redirecting bsAb, even in an interferonresistant tumor. The combination therapy was more effective than eitheragent added alone. Controls with mice treated with TF12 bsAb alone or incombination with interferon-α showed little effect on tumor growth ormortality, compared to untreated animals.

Example 6 In Vivo Therapeutic Use of Antibody-Drug Conjugates (ADCs) inPreclinical Models of Human Pancreatic or Colon Carcinoma

CL2A-SN-38-antibody conjugates were prepared as previously described(see, e.g., U.S. Pat. Nos. 7,999,083 and 8,080,250). Immune-compromisedathymic nude mice (female), bearing subcutaneous human pancreatic orcolon tumor xenografts were treated with either specific CL2A-SN-38conjugate or control conjugate or were left untreated. The therapeuticefficacies of the specific conjugates were observed. In a Capan 1pancreatic tumor model, specific CL2A-SN-38 conjugates of hRS7(anti-TROP2), hPAM4 (anti-MUC5ac), and hMN-14 (anti-CEACAM5) antibodiesshowed better efficacies than control hA20-CL2A-SN-38 conjugate(anti-CD20) and untreated control (not shown). Similarly in a BXPC3model of human pancreatic cancer, the specific hRS7-CL2A-SN-38 showedbetter therapeutic efficacy than control treatments (not shown).Likewise, in an aggressive LS174T model of human colon carcinoma,treatment with specific hMN-14-CL2A-SN-38 was more efficacious thannon-treatment (not shown).

Example 7 In Vivo Therapy of Lung Metastases of GW-39 Human ColonicTumors in Nude Mice Using ADC hMN-14-[CL2-SN-38], IMMU-130

A lung metastatic model of colonic carcinoma was established in nudemice by i.v. injection of GW-39 human colonic tumor suspension, andtherapy was initiated 14 days later. Specific anti-CEACAM5 antibodyconjugate, hMN14-CL2-SN-38, as well as nontargeting anti-CD22 MAbcontrol conjugate, hLL2-CL2-SN-38 and equidose mixtures of hMN14 andSN-38 were injected at a dose schedule of q4d×8, using different doses.Selective therapeutic effects were observed with the hMN-14 ADC (notshown). At a dosage of 250 μg, the mice treated with hMN14-CL2-SN-38showed a median survival of greater than 107 days. Mice treated with thecontrol conjugated antibody hLL2-CL2-SN-38, which does not specificallytarget lung cancer cells, showed median survival of 77 days, while micetreated with unconjugated hMN14 IgG and free SN-38 showed a mediansurvival of 45 days, comparable to the untreated saline control of 43.5days. A significant and surprising increase in effectiveness of theconjugated, cancer cell targeted antibody-SN-38 conjugate, which wassubstantially more effective than unconjugated antibody and freechemotherapeutic agent alone, was clearly seen (not shown). Thedose-responsiveness of therapeutic effect of conjugated antibody wasalso observed (not shown). These results demonstrate the clearsuperiority of the SN-38-antibody conjugate compared to the combinedeffect of both unconjugated antibody and free SN-38 in the same in vivohuman lung cancer system.

Example 8 Use of ADC (IMMU-132 or hRS7-SN-38) to TreatTherapy-Refractive Metastatic Colonic Cancer (mCRC)

The patient was a 62-year-old woman with mCRC who originally presentedwith metastatic disease in January 2012. She had laparoscopic ilealtransverse colectomy as the first therapy a couple of weeks afterdiagnosis, and then received 4 cycles of FOLFOX (leucovorin,5-fluorouracil, oxaliplatin) chemotherapy in a neoadjuvant setting priorto right hepatectomy in March 2012 for removal of metastatic lesions inthe right lobe of the liver. This was followed by an adjuvant FOLFOXregimen that resumed in June, 2012, for a total of 12 cycles of FOLFOX.In August, oxaliplatin was dropped from the regimen due to worseningneurotoxicity. Her last cycle of 5-FU was on Sep. 25, 2012.

CT done in January 2013 showed metastases to liver. She was thenassessed as a good candidate for enrollment to IMMU-132 (hRS7-SN-38)investigational study. Comorbidities in her medical history includeasthma, diabetes mellitus, hypertension, hypercholesteremia, heartmurmur, hiatal hernia, hypothyroidism, carpel tunnel syndrome, glaucoma,depression, restless leg syndrome, and neuropathy. Her surgical historyincludes tubo-ligation (1975), thyroidectomy (1983), cholescystectomy(2001), carpel tunnel release (2008), and glaucoma surgery.

At the time of entry into this therapy, her target lesion was a 3.1-cmtumor in the left lobe of the liver. Non-target lesions included severalhypo-attenuated masses in the liver. Her baseline CEA was 781 ng/mL.

IMMU-132 was given on a once-weekly schedule by infusion for 2consecutive weeks, then a rest of one week, this constituting atreatment cycle. These cycles were repeated as tolerated. The firstinfusion of IMMU-132 (8 mg/kg) was started on Feb. 15, 2013, andcompleted without notable events. She experienced nausea (Grade 2) andfatigue (Grade 2) during the course of the first cycle and has beencontinuing the treatment since then without major adverse events. Shereported alopecia and constipation in March 2013. The first responseassessment done (after 6 doses) on Apr. 8, 2013 showed a shrinkage oftarget lesion by 29% by computed tomography (CT). Her CEA leveldecreased to 230 ng/mL on Mar. 25, 2013. In the second responseassessment (after 10 doses) on May 23, 2013, the target lesion shrank by39%, thus constituting a partial response by RECIST criteria. She hasbeen continuing treatment, receiving 6 cycles constituting 12 doses ofhRS7-SN-38 (IMMU-132) at 8 mg/kg. Her overall health and clinicalsymptoms improved considerably since starting this investigationaltreatment.

Example 9 ADC Therapy with IMMU-132 for Metastatic Solid Cancers

IMMU-132 is an ADC comprising the active metabolite of CPT-11, SN-38,conjugated by a pH-sensitive linker (average drug-antibody ratio=7.6) tothe hRS7 anti-Trop-2 humanized monoclonal antibody, which exhibits rapidinternalization when bound to Trop-2. IMMU-132 targets Trop-2, a type Itransmembrane protein expressed in high prevalence and specificity bymany carcinomas. This Example reports a Phase I clinical trial of 25patients with different metastatic cancers (pancreatic, 7;triple-negative breast [TNBC], 4; colorectal [CRC], 3; gastric, 3,esophageal, prostatic, ovarian, non-small-cell lung, small-cell lung[SCLC], renal, tonsillar, urinary bladder, 1 each) after failing amedian of 3 prior treatments (some including topoisomerase-I and -IIinhibiting drugs).

IMMU-132 was administered in repeated 21-day cycles, with each treatmentgiven on days 1 and 8. Dosing started at 8 mg/kg/dose (i.e., 16mg/kg/cycle), and escalated to 18 mg/kg before encounteringdose-limiting neutropenia, in a 3+3 trial design. Fatigue, alopecia, andoccasional mild to moderate diarrhea were some of the more commonnon-hematological toxicities, with 2 patients also reporting a rash.Over 80% of 24 assessable patients had stable disease or tumor shrinkage(SD and PR) among the various metastatic cancers as best response by CT.Three patients (CRC, TNBC, SCLC) have PRs by RECIST; median TTP for allpatients, excluding those with pancreatic cancer, is >18 weeks.Neutropenia has been controlled by dose reduction to 8-10 mg/kg/dose(16-20 mg/kg/cycle).

Immunohistochemistry showed strong expression of Trop-2 in most archivedpatient tumors, but it is not detected in serum. Correspondingreductions in blood tumor marker titers (e.g., CEA, CA19-9) reflectedtumor responses. No anti-antibody or anti-SN-38 antibodies have beendetected despite repeated dosing. Peak and trough assessments ofIMMU-132 concentrations in the serum show that the conjugate clearscompletely within 7 days, an expected finding based on in vitro studiesshowing 50% of the SN-38 is released in the serum every day. Theseresults indicate that this novel ADC, given in doses ranging from 16-24mg/kg per cycle, shows a high therapeutic index in diverse metastaticsolid cancers.

Example 10 IMMU-130, an SN-38 ADC that Targets CEACAM5, isTherapeutically Active in Metastatic Colorectal Cancer (mCRC)

IMMU-130, an ADC of SN-38 conjugated by a pH-sensitive linker (7.6average drug-antibody ratio) to the humanized anti-CEACAM5 antibody(labetuzumab), is completing two Phase I trials. In both, eligiblepatients with advanced mCRC were required to have failed/relapsedstandard treatments, one being the topoisomerase-I inhibiting drug,CPT-11 (irinotecan), and an elevated plasma CEA (>5 ng/mL).

IMMU-130 was administered every 14 days (EOW) at doses starting from 2.0mg/kg in the first protocol (IMMU-130-01). Febrile neutropenia occurredin 2 of 3 patients at 24 mg/kg; otherwise at ≤16 mg/kg, neutropenia(≥Grade 2) was observed in 7 patients, with one also experiencingthrombocytopenia. One patient [of 8 who received ≥4 doses (2 cycles)]showed a 40.6% decrease in liver (starting at 7 cm) and lung targetlesions (PR by RECIST) for 4.7 months, with no major toxicity,tolerating a total of 18 doses at 16 mg/kg. The study is continuing at12 mg/kg EOW.

Since SN-38 is most effective in S-phase cells, a more protractedexposure could improve efficacy. Thus, in a second Phase I trial(IMMU-130-02), dosing was intensified to twice-weekly, starting at 6mg/kg/dose for 2 weeks (4 doses) with 1 week off, as a treatment cycle,in a 3+3 trial design. Neutropenia and manageable diarrhea were themajor side effects, until dose reduction to 4.0 mg/kg twice-weekly, withearly results indicating multiple cycles are well-tolerated. Currently,tumor shrinkage occurred in 3 patients, with 1 in continuing PR (−46%)by RECIST, among 6 patients who completed ≥4 doses (1 cycle). In bothtrials, CEA blood titers correlated with tumor response, and high levelsdid not interfere with therapy. There have been no anti-antibody oranti-SN-38 antibody reactions, based on ELISA tests. In each study, theADC was cleared by 50% within the first 24 h, which is much longerexposure than with typical doses of the parental molecule, CPT-11. Theseresults indicate that this novel ADC, given in different regimensaveraging ˜16-24 mg/kg/cycle, shows a high therapeutic index in advancedmCRC patients. Since CEACAM5 has elevated expression in breast and lungcancers, as well as other epithelial tumors, it may be a useful targetin other cancers as well.

Example 11 Antitumor Activity of Checkpoint Inhibitor Antibody Alone orCombined with Anti-Trop-2×Anti-CD3 bsAb, IFN-α or Anti-Trop-2 ADC

To determine if the antitumor activity of the exemplary checkpointinhibitor antibody, ipilimumab (anti-CTLA4) is synergistic with orinhibited by the addition of other therapeutic agents, CTLA4 mAb isevaluated alone or in combination with the exemplary T-cell redirectingbsAb (E1)-3s, with interferon-α (PEGINTERFERON®), or with the exemplaryADC hRS7-SN-38 (IMMU-132) in murine tumor models. M109 lung carcinoma,SA1N fibrosarcoma, and CT26 colon carcinoma models are chosen based ondifferent sensitivity to the various agents and CTLA4 blockade. Human Tcells are co-administered with the antibodies.

All compounds are tested at their optimal dose and schedule. When usedin combination, CTLA4 mAb is initiated one day after the first dose ofIMMU-132, (E1)-3s or interferon-α. Percent tumor growth inhibition andnumber of days to reach target tumor size are used to evaluate efficacy.Antitumor activity is scored as: complete regression (CR; non-palpabletumor) or partial regression (PR; 50% reduction in tumor volume).Synergy is defined as antitumor activity significantly superior (p<0.05)to the activity of monotherapy with each agent.

In the SA1N fibrosarcoma tumor model, which is sensitive to CTLA4blockade and modestly sensitive to (E1)-3s, interferon-α, and IMMU-132,borderline synergy is evident with the combination of CTLA4 mAb and(E1)-3s, whereas no effect is observed with interferon-α. IMMU-132monotherapy does not produce significant SA1N antitumor activity.However, combining IMMU-132 with CTLA4 mAb results in synergy. In theM109 lung metastasis model and CT26 colon carcinoma model, synergy isdetected for CTLA4 mAb combined with each of IMMU-132, (E1)-3s andinterferon-α.

In summary, addition of CTLA4 mAb to interferon-α, IMMU-132, or (E1)-3sresults in model-dependent synergistic activities. Synergy is observedregardless of the immunogenicity of the tumor and only when at least oneof the therapies is active. All combination regimens are well-toleratedand the combination therapies do not appear to inhibit CTLA4 mAbactivity. Synergy is observed in tumors unresponsive to CTLA4 mAb alone,suggesting that the other therapeutic agents might induce immunogeniccell death.

Example 12 Combination Therapy with Anti-Trop-2 ADC (IMMU-132) andInterferon-α (PEGINTERFERON®) to Treat Refractory, Metastatic, Non-SmallCell Lung Cancer

The patient is a 60-year-old man diagnosed with non-small cell lungcancer. The patient is given chemotherapy regimens of carboplatin,bevacizumab for 6 months and shows a response, and then afterprogressing, receives further courses of chemotherapy with carboplatin,etoposide, TAXOTERE®, gemcitabine over the next 2 years, with occasionalresponses lasting no more than 2 months. The patient then presents witha left mediastinal mass measuring 6.5×4 cm and pleural effusion.

After signing informed consent, the patient is given IMMU-132 at a doseof 18 mg/kg every other week. After the first week of treatment, thepatient is given combination therapy with IMMU-132 and PEGINTERFERON®.During the first two injections, brief periods of neutropenia anddiarrhea are experienced, with 4 bowel movements within 4 hours, butthese resolve or respond to symptomatic medications within 2 days. Aftera total of 6 infusions of IMMU-132 and 5 infusions of PEGINTERFERON®, CTevaluation of the index lesion shows a 22% reduction, just below apartial response but definite tumor shrinkage. The patient continueswith this therapy for another two months, when a partial response of 45%tumor shrinkage of the sum of the diameters of the index lesion is notedby CT, thus constituting a partial response by RECIST criteria. Thecombination therapy appears to provide a synergistic response, comparedto the two agents administered separately.

Example 13 Combination Therapy with ADC (IMMU-130) and T-CellRedirecting bsAb (MT100) to Treat Advanced Colonic Cancer

The patient is a 75-year-old woman initially diagnosed with metastaticcolonic cancer (Stage IV). She has a right partial hemicolectomy andresection of her small intestine and then receives FOLFOX,FOLFOX+bevacizumab, FOLFIRI+ramucirumab, and FOLFIRI+cetuximab therapiesfor a year and a half, when she shows progression of disease, withspread of disease to the posterior cul-de-sac, omentum, with ascites inher pelvis and a pleural effusion on the right side of her chest cavity.Her baseline CEA titer just before this therapy is 15 ng/mL. She isgiven 6 mg/kg IMMU-130 (anti-CEACAM5-SN-38) twice weekly for 2consecutive weeks, and then one week rest (3-week cycle). After thefirst cycle, the patient is given combination therapy with IMMU-132 andthe leukocyte redirecting bsAb MT110, which is administered bycontinuous infusion on the same 3-week cycle. After 5 cycles, which aretolerated very well, without any major hematological ornon-hematological toxicities, her plasma CEA titer shrinks modestly to1.3 ng/mL, but at the 8-week evaluation she shows a 21% shrinkage of theindex tumor lesions, which increases to a 27% shrinkage at 13 weeks.Surprisingly, the patient's ascites and pleural effusion both decrease(with the latter disappearing) at this time, thus improving thepatient's overall status remarkably. The combination therapy appears toprovide a synergistic response, compared to the two agents administeredseparately.

Example 14 Combination Therapy with ADC (IMMU-130), Anti-Trop-2×Anti-CD3bsAb ((E1)-3s) and Interferon-α to Treat Gastric Cancer Patient withStage IV Metastatic Disease

The patient is a 52-year-old male who sought medical attention becauseof gastric discomfort and pain related to eating for about 6 years, andwith weight loss during the past 12 months. Palpation of the stomacharea reveals a firm lump which is then gastroscoped, revealing anulcerous mass at the lower part of his stomach. This is biopsied anddiagnosed as a gastric adenocarcinoma. Laboratory testing reveals nospecific abnormal changes, except that liver function tests, LDH, andCEA are elevated, the latter being 10.2 ng/mL. The patent then undergoesa total-body PET scan, which discloses, in addition to the gastrictumor, metastatic disease in the left axilla and in the right lobe ofthe liver (2 small metastases). The patient has his gastric tumorresected, and then has baseline CT measurements of his metastatictumors. Four weeks after surgery, he receives 3 courses of combinationchemotherapy consisting of a regimen of cisplatin and 5-fluorouracil(CF), but does not tolerate this well, so is switched to treatment withdocetaxel. It appears that the disease is stabilized for about 4 months,based on CT scans, but then the patient's complaints of further weightloss, abdominal pain, loss of appetite, and extreme fatigue causerepeated CT studies, which show increase in size of the metastases by asum of 20% and a suspicious lesion at the site of the original gastricresection.

The patient is then given experimental therapy with IMMU-130(anti-CEACAM5-SN-38) on a weekly schedule of 8 mg/kg. After the firstweek, combination therapy with IMMU-130, (E1)-3s and interferon-α isinitiated. The patient exhibits no evidence of diarrhea or neutropeniaover the following 4 weeks. The patient then undergoes a CT study tomeasure his metastatic tumor sizes and to view the original area ofgastric resection. The radiologist measures, according to RECISTcriteria, a decrease of the sum of the metastatic lesions, compared tobaseline prior to therapy, of 23%. There does not seem to be any clearlesion in the area of the original gastric resection. The patient's CEAtiter at this time is 7.2 ng/mL, which is much reduced from the baselinevalue of 14.5 ng/mL. The patient continues on weekly combinationtherapy, and after a total of 13 infusions, his CT studies show that oneliver metastasis has disappeared and the sum of all metastatic lesionsis decreased by 41%, constituting a partial response by RECIST. Thepatient's general condition improves and he resumes his usual activitieswhile continuing to receive maintenance therapy every third week. At thelast measurement of blood CEA, the value is 4.8 ng/mL, which is withinthe normal range for a smoker, which is the case for this patient.

Example 15 General Techniques for Dock-and-Lock®

The general techniques discussed below may be used to generate DNL®complexes with AD or DDD moieties attached to any antibodies orantigen-binding antibody fragments, using the disclosed methdods andcompositions.

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD or Fab-ADexpression vectors.

To generate Fab-DDD expression vectors, the coding sequences for thehinge, CH2 and CH3 domains of the heavy chain were replaced with asequence encoding the first 4 residues of the hinge, a 14 residue linkerand a DDD moiety, such as the first 44 residues of human RIIα (referredto as DDD1, SEQ ID NO:1). To generate Fab-AD expression vectors, thesequences for the hinge, CH2 and CH3 domains of IgG were replaced with asequence encoding the first 4 residues of the hinge, a 15 residue linkerand an AD moiety, such as a 17 residue synthetic AD called AKAP-IS(referred to as AD1, SEQ ID NO:3), which was generated usingbioinformatics and peptide array technology and shown to bind RIIαdimers with a very high affinity (0.4 nM). See Alto, et al. Proc. Natl.Acad. Sci., U.S.A (2003), 100:4445-50. Two shuttle vectors were designedto facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD1 orFab-AD1 expression vectors, as described below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC, SEQ ID NO:102) followed byfour glycines and a serine, with the final two codons (GS) comprising aBam HI restriction site. The 410 bp PCR amplimer was cloned into thePGEMT® PCR cloning vector (PROMEGA®, Inc.) and clones were screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of DDD1 preceded by 11 residues of the linker peptide, with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 103) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 bp DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplimer was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 104) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective PGEMT® shuttle vector.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 (SEQ ID NO:2) appended to the carboxyl terminus of theFd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker. Thefusion protein secreted is composed of two identical copies of hMN-14Fab held together by non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair to C-DDD2-Fab-hMN-14.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2 (SEQID NO:4) appended to the carboxyl terminal end of the CH1 domain via a14 amino acid residue Gly/Ser peptide linker. AD2 has one cysteineresidue preceding and another one following the anchor domain sequenceof AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Generation of TF2 DNL® Construct

A trimeric DNL® construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Production of TF10 DNL® Construct

A similar protocol was used to generate a trimeric TF10 DNL® construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The TF10 bispecific ([hPAM4]₂×h679) antibody was producedusing the method disclosed for production of the (anti CEA)₂×anti HSGbsAb TF2, as described above. The TF10 construct bears two humanizedPAM4 Fabs and one humanized 679 Fab.

The two fusion proteins (hPAM4-DDD2 and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD2. The reaction mixture was incubated at room temperaturefor 24 hours under mild reducing conditions using 1 mM reducedglutathione. Following reduction, the reaction was completed by mildoxidation using 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP291-affigel resin, which binds with highspecificity to the h679 Fab.

Example 16 Production of AD- and DDD-Linked Fab and IgG Fusion Proteinsfrom Multiple Antibodies

Using the techniques described in the preceding Example, the IgG and Fabfusion proteins shown in Table 11 were constructed and incorporated intoDNL® constructs. The fusion proteins retained the antigen-bindingcharacteristics of the parent antibodies and the DNL® constructsexhibited the antigen-binding activities of the incorporated antibodiesor antibody fragments.

TABLE 11 Fusion proteins comprising IgG or Fab Binding Fusion ProteinSpecificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSG C-(AD)₂-Fab-h679 HSGC-AD2-Fab-h734 Indium- DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DRC-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1 IGF-1RC-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

Example 17 Use of NK-Targeted Leukocyte-Redirecting bsAbs

The use of bsAbs to retarget leukocytes is not limited to antibodiesagainst T cells. In alternative embodiments, bsAbs that bind tomonocytes, NK cells or neutrophils may also be used for retargetingpurposes.

CD16 is an activating low-affinity Fc-γ receptor for IgG, which ishighly expressed by the CD56^(dim) subset of NK cells (Gleason et al.,2012, Mol Cancer Ther 11:2674-84). In addition to their use in NK cellretargeting, bsAbs comprising an anti-CD16 antibody component have theability to activate NK-mediated cytotoxicity through direct signaling ofCD16, inducing directed secretion of lytic granules and target celldeath (Gleason et al., 2012).

A CD16/CD19 bispecific killer cell engager (BiKE) and a CD16/CD19/CD22trispecific killer cell engager (TriKe) are prepared according to(Gleason et al., 2012, Mol Cancer Ther 11:2674-84), using DNA shufflingand ligation techniques as previously reported (Vallera et al., 2005,Clin Cancer Res 11:3879-88). The expressed BiKE and TriKE are purifiedby sequential ion exchange and size-exclusion column chromatography.Resting PBMCs are exposed to primary ALL and CLL tumor cells in thepresence of CD16/CD19 BiKE or CD16/CD19/CD22 TriKE (10 μg/mL). Asignificant increase in cytotoxicity to tumor cells is observed in thepresence of the BiKE or TriKE, compared to cells without retargetingantibody. No effect is observed on tumor cells exposed to BiKE or TriKEin the absence of PBMCs. The TriKE has a greater effect on tumor celltoxicity relative to the BiKE, indicating that binding to an additionaltumor cell antigen may enhance the retargeting effect. Similar resultsare obtained using purified NK cells instead of PBMCs.

A CD16/CD33 BiKE is prepared as disclosed in Wiernik et al. (2013, ClinCancer Res 19:3844-55. The BiKE is administered to nude mice injectedwith human HL60 promyelocytic leukemia xenograft cells, co-administeredwith human PBMCs. The BiKE treated mice show a decreased mortality andtumor growth rate compared to mice treated with control bsAbs. Additionof an anti-CD33-SN-38 ADC further enhances the cytotoxic effect of theBiKE.

Example 18 Trivalent Antibodies for Therapeutic Use

A trivalent, trispecific cell targeting construct is made as describedin patent EP1309795B1 comprising: (i) chimerizing or humanizing a mouseanti-CD16 mab as described in U.S. Pat. No. 618,728 from which the Fabof claim 1 of EP1309795 is derived; (ii) constructing a single chainantibody comprised of the Fv of the humanized anti-Trop-2 antibodydescribed in U.S. Pat. No. 7,238,785, and joining the scFv by a linkerto the carboxyl terminal of the light chain of the anti-CD16 Fab of (i);and (iii) constructing a single chain of the Fv of the humanizedanti-CD19 described in U.S. Pat. No. 8,486,395 and joining the scFv by alinker to the carboxyl terminal of the CH1 of the anti-CD16 Fab of (ii).

The trivalent construct is administered to a subject with metastaticpancreatic cancer, in combination with IMMU-132. A partial response isobserved and the tumor shows a regression in size that lasts for 12months.

Example 19 Anti-Trop-2×Anti-CD3 Bispecific Antibody

A bispecific antibody (bsAb) was produced as a tandem single-chainvariable fragment (scFv) for redirecting T cells via CD3 binding totumor cells, particularly carcinomas, via Trop-2 targeting. Trop-2 is atumor-associated antigen (TAA) that could be highly effective fortargeting various epithelial cancers. However, it has yet to beinvestigated in any bsAb format for T-cell-redirected therapy. Trop-2 isa 35 kDa transmembrane glycoprotein that is overexpressed relative tonormal tissues in a variety of human cancers, including pancreatic andgastric carcinomas, where increased expression correlates with poorprognosis (Fong et al., 2008, Br J Cancer 99:1290-5; Iacobuzio-Donahueet al., 2002, Am J Pathol 160:1239-49; Kapoor, 2013, Tumour Biol34:1967-8; Muhlmann et al., 2009, J Clin Pathol 62:152-8; Stein et al.,1993, Int J Cancer 55:938-46; Stein et al., 1993, Int J Cancer55:938-46). Variable domains (VH and VK) derived from hRS7, thehumanized version of the original murine anti-Trop-2 mAb, RS7, werecombined with the variable domains of the murine anti-CD3 mAb, Okt3, togenerate the E1-3 bsAb.

Construction of a Plasmid Vector for Expression of E1-3 in MammalianCells

A double stranded DNA sequence (SEQ ID NO:106) was synthesized andassembled into the pUC57 plasmid vector. SEQ ID NO:106 was excised frompUC57 by digestion with Xba I and Eag I restriction endonucleases, andligated into the pdHL2 mammalian expression vector, which was preparedby digestion with the same enzymes. The coding sequence directs thesynthesis of a single polypeptide (SEQ ID NO:107) comprising a leaderpeptide, hRS7VK (SEQ ID NO:108), L1 (SEQ ID NO:109), hRS7VH (SEQ IDNO:110), L2 (SEQ ID NO:111), Okt3VH (SEQ ID NO:112), L3 (SEQ ID NO:113),Okt3VK (SEQ ID NO:114), and 6-His (SEQ ID NO:105). A schematicrepresentation of the tandem scFv E1-3 is shown in FIG. 22.

Synthetic DNA sequence comprising E1-3 insert (SEQ ID NO: 106)tctagacacaggccgccatcatgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgacattcagctgacccagtctccatcctccctgtctgcatctgtaggagacagagtcagcatcacctgcaaggccagtcaggatgtgagtattgctgtagcctggtatcagcagaaaccagggaaagcccctaagctcctgatctactcggcatcctaccggtacactggagtccctgataggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcagtttattactgtcagcaacattatattactccgctcacgttcggtgctgggaccaaggtggagatcaaaggtggaggagggtccggtggaggagggtctggtggaggagggagccaggtccagctgcagcaatctgggtctgagttgaagaagcctggggcctcagtgaaggtttcctgcaaggcttctggatacaccttcacaaactatggaatgaactgggtgaagcaggcccctggacaagggcttaaatggatgggctggataaacacctacactggagagccaacatatactgatgacttcaagggacggtttgccttctccttggacacctctgtcagcacggcatatctccagatcagcagcctaaaggctgacgacactgccgtgtatttctgtgcaagaggggggttcggtagtagctactggtacttcgatgtctggggccaagggtccctggtcaccgtctcctcaggtggcggagggtccgatatcaagctgcagcagtctggagcagagctcgctcgaccaggagctagtgtgaagatgtcatgtaaaacaagtggctatactttcacccggtacactatgcactgggtcaagcagcgcccaggacagggtctggaatggatcggctacattaaccccagcaggggatataccaactacaatcagaagttcaaggataaagccaccctgactaccgacaagtcctctagtacagcttatatgcagctgtcaagcctcacttccgaggactctgcagtgtattactgcgccagatattacgacgatcattattgtctggattactggggccagggaacaactctcacagtgtcctctgtcgaaggtggcagtggagggtcaggtggcagcggagggtccggtggagtggacgatatccagctgacccagtctcctgccattatgagcgcttccccaggcgagaaggtgacaatgacttgccgggccagttcaagcgtcagctatatgaattggtatcagcagaagtctggaaccagtcctaaacgatggatctatgacacatctaaagtggcaagcggggtcccatacaggttctctgggagtggttcaggcactagctattccctgaccatttcctctatggaggccgaagatgcagccacctattactgtcagcagtggagttcaaatccactcaccttcggagcaggcactaaactggaactcaagcaccaccaccaccaccactaaggcggccg Deduced amino acid sequence of E1-3(SEQ ID NO: 107) DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKHHHHHHAmino acid sequence of hRS7 VK (SEQ ID NO: 108)DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGA GTKVEIKAmino acid sequence of linker L1 (SEQ ID NO: 109) GGGGSGGGGSGGGGSAmino acid sequence hRS7 VH (SEQ ID NO: 110)QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGG FGSSYWYFDVWGQGSLVTVSSAmino acid sequence of linker L2 (SEQ ID NO: 111) GGGGSAmino acid sequence of Okt3 VH (SEQ ID NO: 112)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSSAmino acid sequence of linker L3 (SEQ ID NO: 113) VEGGSGGSGGSGGSGGVDAmino acid sequence of Okt3 VK (SEQ ID NO: 114)DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAG TKLE

Development of a Stable Production Clone in SpESF Myeloma Cells

The E1-3-pdHL2 vector was linearized by digestion with Sal I restrictionendonuclease and 30 μg was used to stably transfect 1×10⁷ SpESFX myelomacells (Rossi et al., 2011, Biotechnol Prog 27:766-75) by electroporationusing two pulses at 850 V and 10 μF. Selection and production media wassupplemented with 0.2 μM methotrexate (MTX). Transfectant clones wereselected in 96-well tissue culture plates and screened for E1-3expression by ELISA using Ni-NTA 96-well plates. The E1-3 protein waspurified from the culture broth of roller bottle cultures by immobilizedmetal affinity chromatography (IMAC) using Nickel-SEPHAROSE® resin,followed by size exclusion high performance liquid chromatography(SE-HPLC). The purified product resolved as a single SE-HPLC peak (notshown) and a single polypeptide band by SDS-PAGE (not shown), withrelative mobilities consistent with its calculated molecular size of53,423 Da.

Example 20 Redirected T Cell Killing of Trop-2-Expressing Solid TumorCells Ex Vivo

Peripheral blood mononuclear cells (PBMCs) were prepared from the buffycoat of blood specimens of two healthy donors (Blood Center of NJ), andused for the isolation of CD8⁺ T cells (Miltenyi). Capan-1 (pancreaticcancer, 157,000 Trop-2/cell), BxPC3 (pancreatic cancer, 500,000Trop-2/cell) and NCI-N87 (gastric cancer, 247,000 Trop-2/cell) celllines (ATCC) were used as target cells expressing low-, high- andmid-levels of Trop-2. BxPC3 and NCI-N87 were maintained in RPMI1640media supplemented with 10% FBS, while Capan-1 cells were maintained in20% FBS/RPMI1640. CD8⁺ T cells (1.2×10⁵ cells/well) were combined withtarget cells (2×10⁴ cells/well) at a 6:1 ratio in 96-well tissue cultureplates. Titrations of E1-3 and (E1)-3s were added to the assay plates.Following a 48-hour incubation at 37° C., plates were washed twice withPBS to remove the T cells, and then 150 μL of fresh media supplementedwith 30% MTS reagent (CELLTITER 96® Aqueous One Solution, Promega) wasadded to each well. Absorbance at 490 nm was measured after 1-2 h at 37°C. with an ENVISION plate Reader®.

The in vitro potency of the E1-3 bispecific antibody was compared withthat of the equivalent DNL construct, (E1)-3s, in threeTrop-2-expressing cell lines (BxPC3, Capan-1 and NCI-N87) using T cellsfrom three donors for each cell line (FIG. 23). Based on the IC₅₀ values(Table 12), E1-3 is at least 5-fold more potent than (E1)-3s in allthree cell lines, whose relative sensitivity to E1-3 appears tocorrelate with the Trop-2-antigen density, when compared with T cellsfrom the same donor. However, potency was varied among the donor T cellsused. In vitro, E1-3 mediated a highly potent T-cell lysis of BxPC3[IC₅₀=0.09(±0.04) pM], Capan-1 [IC₅₀=1.2(±1.1) pM] and NCI-N87[IC₅₀=1.2(±1.2) pM] target cells.

TABLE 12 IC₅₀ values for ex vivo T cell redirected killing of Trop-2⁺cancer cell lines with E1-3 and (E1)-3s. BxPC3 Capan-1 NCI-N87500,000/cell 157,000/cell 247,000/cell Trop-2 Donor 1 Donor 2 Donor 3Donor 1 Donor 2 Donor 4 Donor 1 Donor 2 Donor 5 E1-3 0.12 0.10 0.05 0.582.7 0.47 0.29 0.76 2.50 (E1)-3s 1.06 0.56 0.32 35.6 248 8.51 6.76 34 NA*IC₅₀ values = pM concentration resulting in 50% killing. *Did notachieve 50% killing. Donors 1 and 2 were the same for each donor. Donors3, 4 and 5 were independent donors.

Example 21 In Vivo Therapy of Solid Tumors with E1-3 Vs. (E1)-3s

Female 4-8-week old NOD/SCID mice were administered subcutaneousinjections of a mixture of PBMCs and NCI-N87 (2:1) mixed with an equalvolume of MATRIGEL®. Therapy consisted of i.v. injections of 50 μg ofE1-3 on days 1 and 4, or daily injections with 47 μg of (E1)-3s on days1 through 5. The untreated group received the mixture of NCI-N87 andPBMCs without bsAb. Tumor volume (TV) was determined twice weekly bymeasurements in two dimensions using calipers, with volumes defined as:L×W²/2, where L is the longest dimension of the tumor and W the shortest(FIG. 24). Statistical analysis of tumor growth was based on area underthe curve (AUC). Profiles of individual tumor growth were obtainedthrough linear-curve modeling. An F-test was employed to determineequality of variance between groups prior to statistical analysis ofgrowth curves. A Critical Z test on the survival data identified anyoutliers within a given treatment group with P<0.05 censored from thefinal data analysis. A two-tailed t-test was used to assess statisticalsignificance between the various treatment groups and controls, exceptfor the untreated control, where a one-tailed t-test was used.Additionally, efficacy was determined by log-rank using Prism softwareon Kaplan-Meier curves using survival surrogate endpoints as time fortumor progression (TTP) to 1.0 cm3. Significance was considered atP≤0.05 for all comparisons.

Both E1-3 (P) and (E1)-3s delayed growth of NCI-N87 tumors significantly(P≤0.001; AUC_(day 25)) (FIG. 24). The E1-3 was superior to (E1)-3s(P=0.0324, AUC_(day36)) (FIG. 24). In vivo, two 50-μg doses of E1-3given three days apart cured all of the mice (N=8) bearing NCI-N87xenografts (P=0.0005; Log-Rank). Tumors in the control group (PBMCsonly) reached the endpoint (TV>1 cm³) at 39.5 days. All mice weretumor-free in the E1-3 group after 78 days.

Example 22 Trogocytosis Induced by Anti-CD3×Anti-Trop-2 BispecificAntibodies

Trop-2 has limited presence on normal tissues but is highly expressed indiverse epithelial cancers. As discussed in the Examples above, (E1)-3sis a T-cell-redirecting trivalent bispecific antibody (bsAb) DNL®complex, comprising an anti-CD3 scFv covalently linked to a stabilizeddimer of a Trop-2-targeting Fab. We show herein for the first time thatbsAb-mediated bidirectional trogocytosis occurs between target cells andT cells and involves formation of immunologic synapses.

Methods

BxPC3 cells were detached with trypsin (which does not affect Trop-2)and mixed with purified T cells. Cell mixtures were treated with 0.1nmol/L bsAbs at 37° C. for 1 hour. Cells were stained with either: (i)anti-Trop-2 MABC518 followed by GAM-FITC, or (ii) anti-Trop-2-PE cloneMR54 and anti-CD4-APC. Single BxPC3 and T cells were gated from cellconjugates by forward and side scattering, as well as Trop-2 and CD4fluorescence.

Results

(E1)-3s induces the formation of immunologic synapses between T cellsand target cells. This was shown using Capan-1 pancreatic carcinomacells (Rossi et al., 2013, MAbs 6:381-91). Here, addition of 0.1 μg/mL(E1)-3s to a mixture of purified CD8⁺ T cells and NCI-N87 gastriccarcinoma cells, which were membrane-labeled with red and greenfluorescence, respectively, resulted in the formation of conjugatesevident by fluorescence microscopy (not shown). No conjugates wereobserved in the presence of (19)-3s (not shown) or TF12 (not shown),which bind only T cells or NCI-N87, respectively. Dunking the slides insaline washed off the vast majority of T cells in wells containing(19)-3s or TF12, whereas many T cells remained bound to the adherentNCI-N87 cells in the wells treated with (E1)-3s.

Treatment of BxPC3 (500,000 Trop-2/cell) and purified T-cell mixtureswith (E1)-3s specifically induced trogocytosis, whereby Trop-2 wastransferred from BxPC3 to T cells (FIG. 25). Whereas (E1)-3s treatmentresulted in 40% Trop-2⁺ T cells, <5% of the T cells were counted in theTrop-2⁺ gate following treatment with control bsAbs binding only Trop-2(TF12) or CD3 [(20)-3s], or with (E1)-3s in the absence of BxPC3 cells.The uptake of Trop-2 by T cells coincided with its reduction on BcPC3cells (FIG. 26). During the short incubation time, the T cells (97.5%live) and BxPC3 (94.5% live) remained at high viability, indicating thatthe T cells acquired the tumor antigens by trogocytosis and not byadhering to membrane fragments of dead cells (not shown). Trogocytosismediated by (E1)-3s was bidirectional, because T-cell membranecomponents were transferred to BxPC3 cells, as demonstrated for CD4(data not shown).

Example 23 Bispecific Anti-CD3×Anti-Trop-2 Antibodies and CytokineRelease

As discussed in the Examples above, we studied the effects ofinterferon-α (IFNα) on (E1)-3s-mediated T-cell killing of human gastricand pancreatic cancer cell lines. T-cell activation, cytokine induction,and cytotoxicity were evaluated ex vivo using peripheral bloodmononuclear cells (PBMC) or T cells with NCI-N87 gastric cancer astarget cells. In the presence of target cells and PBMCs, (E1)-3s did notcause excess cytokine production. When combined with (E1)-3s,peginterferonalfa-2a—which alone did not increase T-cell activation orraise cytokine levels over baseline—increased CD69 expression but didnot significantly increase cytokine induction. IFNα enhanced thetherapeutic efficacy of (E1)-3s without increasing the production ofother cytokines to levels that could induce cytokine release syndrome(CRS). Adjuvant therapy with IFNα, or other cytokines, might beuniversally applicable for enhanced efficacy of T-cell immunotherapy.

Methods

Cytokine release was measured ex vivo using 5×10⁵ cells/0.5 mL/well ofeither NCI-N87, which were allowed to attach overnight, or Raji. Freshlyisolated PBMCs (5×10⁶ cells/0.4 mL/well) were added to each well.Treatments (100 μL, 10×) comprising (19)-3s, 19-3 BiTE, (E1)-3s,peginterferonalfa-2a, or (E1)-3s plus peginterferonalfa-2a were added to0.1 nmol/L for each reagent. Alternatively, titrations ranging from 1pmol/L to 10 nmol/L were used for dose-response studies. Following a20-hour incubation at 37° C. with gentle shaking, the supernatant fluidwas diluted 1:2 (or greater when necessary) and the concentrations ofTNFα, IFNg, IL2, IL6, and IL10 measured using Single-Analyte ELISArraykits (Qiagen), following the manufacturer's protocol.

Results

A Trop-2×CD3 BiTE (or equivalent) was not available for comparison with(E1)-3s. However, the availability of both (19)-3s, which has the same(X)-3s molecular configuration as (E1)-3s, and 19-3 BiTE, which has theidentical amino acid sequence as the CD19×CD3 BiTE, blinatumomab,enabled a head-to-head comparison to evaluate the relativecytokine-inducing potency of the two bsAb formats.

Initially, titrations of (19)-3s and 19-3 BiTE were added to mixtures ofPBMCs (two independent donors), and Raji NHL cells and the levels ofTNFα, IFNγ, and IL6 were measured after 20 hours (not shown). Minimalcytokine levels were detected from PBMCs alone, even with the additionof a bsAb. However, because of a mixed lymphocyte reaction occurringbetween Raji and the donor PBMCs (stronger for donor A), cytokine levelsin untreated cell mixtures were elevated for each of TNFα (200 and 50pg/mL), IFNγ (600 and 200 pg/mL), and IL6 (190 and 220 pg/mL). Thelevels of TNFα and IL6 were increased above those of untreated only at≥1 nmol/L (19)-3s. Apparently, (19)-3s inhibited TNFα and IL6 productionat lower concentrations. In comparison, TNFα and IL6 were elevatedto >1,000 pg/mL at all concentrations of 19-3 BiTE tested (≥1 pmol/L).The levels of IFNγ were not increased significantly by (19)-3s, whereas19-3 BiTE showed a dose-dependent increase to >2,000 pg/mL.

For all further comparisons, agents were tested at 0.1 nmol/L, which isapproximately what has been used in similar studies with BiTE (Brandl etal., Cancer Immunol Immunother 2007, 56:1551-63). We compared the levelsof TNFα, IFNγ, IL2, IL6, and IL10 induced by 0.1 nmol/L (19)-3s or 19-3BiTE from Raji mixed with PBMCs, using 4 different donors (FIG. 27A).With each of the 4 donors, the levels of each of the five cytokines weresignificantly higher with 19-3 BiTE, compared with (19)-3s. The meanTNFα concentration with 19-3 BiTE (2,284±1,483 pg/mL) was 8-fold higher(P=0.0001) than that with (19)-3s (280±188 pg/mL). Treatment with 19-3BiTE, compared with (19)-3s, resulted in levels of IFNγ (3,002±560 pg/mLvs. 416±169 pg/mL), IL2 (13,635±2,601 pg/mL vs. 1,024±598 pg/mL), IL6(981±364 pg/mL vs. 168±96 pg/mL), and IL10 (4,006±2,520 pg/mL vs.493±242 pg/mL) that were 7-, 13-, 6-, and 8-fold higher for 19-3 BiTE,respectively (P<0.0001 for each). These results indicate that the (X)-3sbsAb format is a considerably less potent inducer of cytokine release,compared with the BiTE format.

In general, (E1)-3s in the presence of PBMCs and target cells causedeven less cytokine production than (19)-3s, because there is no mixedlymphocyte reaction to elevate the baseline levels (FIG. 27B). Levelsremained low for the proinflammatory cytokines IFNγ (<100 pg/mL), TNFα(<100 pg/mL), and IL2 (<250 pg/mL) with 4 of 5 donors. IL6 was low (<400pg/mL) in 3 of 5 donors, and moderate (800-1,100 pg/mL) in donors D-2and D-5. Donor D-2 also responded to the (E1)-3s more than the othersfor IFNγ (1,000 pg/mL) and TNFα (190 pg/mL). IL10, an anti-inflammatorycytokine, was significantly (P<0.0001) elevated by (E1)-3s to >1,200pg/mL in 3 of 5 donors. Of note, donor D-2, who had a uniquely potentproinflammatory response, produced relatively low levels of IL10 (230pg/mL) after treatmentwith (E1)-3s. Peginterferonalfa-2a alone did notincrease the level of any cytokine over background. Addition ofpeginterferonalfa-2a to (E1)-3s consistently increased IFNγ(˜1.5-3-fold) over (E1)-3s alone. For the rest of the cytokines, therewas an apparent trend for a moderately increased production with thecombination; however, a consistent effect was not observed.

Example 24 In Vitro Cytotoxicity Induced by BispecificAnti-CD3×Anti-Trop-2 Antibodies

Further studies were performed to examine in vitro cytotoxicity inducedby anti-CD3×anti-Trop-2 bispecific antibodies.

Methods

Freshly-isolated CD8⁺ T cells were incubated for 24 h with 0.1 nMpeginterferonalfa-2a, 0.1 nM 20*-2b, or media only. Treated or untreatedT cells and PKH67 green fluorescent-labeled NCI-N87 cells were combinedat a 5:1 ratio (5×10⁴ target cells and 2.5×10⁵ effector cells/well) in48-well plates containing serial dilutions of (E1)-3s in triplicate.Peginterferonalfa-2a or 20*-2b were maintained at 0.1 nM in theappropriate cell mixtures. Plates were incubated for 48 h at 37° C.Suspension cells were removed and the attached cells were detached withtrypsin-EDTA and combined with the corresponding suspension. Cells werewashed and resuspended in 1% BSA-PBS containing 30,000 COUNTBRIGHT™Absolute Counting Beads (Life Technologies) and 1 μg/mL of 7-AAD. Totallive target cells (7-AAD7PKH67⁺) were counted by flow cytometry. Foreach sample, 8,000 COUNTBRIGHT™ beads were counted as a normalizedreference. The specific lysis (%) was calculated using the formula:[1-(A₁/A₂)]×100, where A₁ and A₂ represent the number of viable targetcells in the test and untreated samples, respectively. Statisticalsignificance (P≤0.05) was determined for IC₅₀ (the concentrationresulting in 50% lysis), EC₅₀ (50% effective concentration) andlysis^(max) (maximal target cell lysis) by F-test on non-linearregression (sigmoidal dose-response) curves with Prism software.

Results

To evaluate redirected T-cell killing of Trop-2-expressing tumor cells,CD8⁺ T cells were mixed with NCI-N87 cells in the presence or absence ofIFN-α2 (0.1 nM peginterferonalfa-2a or 20*-2b) along with titrations of(E1)-3s (FIG. 28). Considerable variability in T-cell potency wasobserved among donors (FIG. 28A, FIG. 28B). With a donor of very activeT cells, (E1)-3s mediated a highly potent (IC₅₀=0.37 pM;lysis^(max)=77.1%) T-cell lysis of NCI-N87 cells, and inclusion ofpeginterferonalfa-2a enhanced its activity, improving the IC₅₀ (0.14 pM;P=0.0001) by more than 2.5 fold and increasing lysis^(max) (84.0%;P<0.0001) (FIG. 28A). NCI-N87 was only weakly sensitive to the directactions of IFN-α (peginterferonalfa-2a IC₅₀=>10 nM, data not shown), andinhibited <10% by 0.1 nM peginterferonalfa-2a in the absence of (E1)-3s.The more potent form of IFNα, 20*-2b, consisting of 4 IFN-α moleculesfused to a bivalent anti-CD20 mAb by DNL®, enhanced the potency of(E1)-3s by more than 7-fold (IC₅₀=0.05 pM; P<0.0001). At 0.1 nM, 20*-2binhibited NCI-N87 by 12.6% in the absence of (E1)-3s. The 20*-2b wasincluded only to show enhanced activity with another (more potent) formIFN-α, and that the effect is not restricted to peginterferonalfa-2a.The anti-CD20 mAb moiety is not functional in this experiment. In asimilar assay using very weak donor T cells, (E1)-3s was considerablyless potent (EC₅₀=39 pM; lysis^(max)=21%); however, addition ofpeginterferonalfa-2a enhanced the potency by >25 fold (EC₅₀=1.4 pM;P=0.0008) (FIG. 28B). Potent (E1)-3s-mediated T-cell killing also wasobserved for the human pancreatic cancer line, BxPC3 (IC₅₀=0.4 pM);however, the effect of adding IFN-α was not evaluated with this cellline (not shown).

Example 25 Dose-Response Curves for T Cell Activation byAnti-CD3×Anti-Trop-2 Bispecific Antibodies

Addition of 0.1 nM peginterferonalfa-2a increased CD69 upregulation on Tcells treated with (E1)-3s moderately, but significantly. For (E1)-3sdose-response experiments measuring % CD69⁺ T cells, the EC₅₀ waslowered from 26 pM to 16 pM (P<0.0001) for CD4⁺ T cells, and from 11 pMto 6 pM (P=0.0204) for CD8⁺ T cells in the presence of IFN-α (FIG. 29A).Peginterferonalfa-2a combined with (E1)-3s resulted in more CD69⁺ cells(FIG. 29B, FIG. 29C, P<0.0001), and also, the activated cells hadsignificantly higher CD69 expression with IFN-α (FIG. 29B, FIG. 29D;MFI=907 vs 726; P<0.0001). Peginterferonalfa-2a induced minimal CD69expression in the absence of (E1)-3s. Likewise, (E1)-3, either alone orin combination with peginterferonalfa-2a, did not activate T cells inthe absence of target cells.

Example 26 Extended In Vivo Survival with (E1)-3s is Augmented withIFN-α

The preliminary data on in vivo survival reported in Example 3 abovewere further extended to as long as 126 days. As shown below, thecombination of (E1)-3s with IFN-α provided the greatest benefit foranimals bearing Trop-2⁺ xenograft tumors.

Methods

Female 4-8-week old NOD/SCID mice (Charles River, Wilmington, Mass.)were injected s.c. with a mixture of 5×10⁶ tumor cells (Capan-1 orNCI-N87) and T cells (2.5×10⁶) combined with an equal volume ofmatrigel. Therapy began 1 h later by i.v. injection, as per the BiTEmethodology (Dreier et al., 2003, J Immunol 170:4397-402). Treatmentregimens, dosages, and number of animals in each experiment aredescribed in the figure legends. Tumor volume was determined twiceweekly by measurements in two dimensions using calipers, with volumesdefined as: L×w²/2, where L is the longest dimension of the tumor and wthe shortest.

Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. An F-test was employed to determine equality ofvariance between groups prior to statistical analysis of growth curves.A Critical Z test on the survival data identified any outliers within agiven treatment group with P≤0.05 censored from the final data analysis.A two-tailed t-test was used to assess statistical significance betweenthe various treatment groups and controls, except for the untreatedcontrol, where a one-tailed t-test was used. Additionally, efficacy wasdetermined by log-rank using Prism software on Kaplan-Meier curves usingsurvival surrogate endpoints as time for tumor progression to 1.0 cm³.Significance was considered at P≤0.05 for all comparisons.

Results

In vivo efficacy with human pancreatic cancer was evaluated with Capan-1xenografts. In the first study, treatment with a combination of (E1)-3sand peginterferonalfa-2a [median survival time (MST)>59 days] wassuperior to all other treatments (P<0.0007, log-rank), including (E1)-3s(MST=50 days) or peginterferonalfa-2a (MST=53 days) alone (FIG. 30A).Even with the omission of T cells, peginterferonalfa-2a extendedsurvival (MST=45 days, P=0.0059 vs saline, log-rank), indicating directaction on the tumor cells. However, peginterferonalfa-2a was moreeffective in the presence of T cells (P=0.0260, AUC), suggestingstimulation of T cells by IFN-α. TF12, which binds target but not Tcells, did not affect tumor growth or survival. A repeat experiment,using T cells from a different donor, confirmed the results of the firststudy (FIG. 30B). The second study continued until all groups reachedtheir MST. As in the initial experiment, the combination of (E1)-3s andpeginterferonalfa-2a (MST=119.5 days) was superior to all other groupsin terms of both tumor growth inhibition and overall survival (P=0.0475vs (E1)-3s alone; P<0.0001 vs all other groups; log-rank). (E1)-3s(MST=68 days) was superior (P=0.0373, AUC over 29 days) topeginterferonalfa-2a with T cells (MST=53 days) and to T cells alone(MST=37.5 days; P=0.0014 log-rank).

For the NCI-N87 gastric cancer xenograft model (FIG. 30C), thecombination of (E1)-3s and peginterferonalfa-2a (MST>88 days) wassuperior to (E1)-3s alone (MST=49 days; P=0.0007, log-rank). Compared tothe control group with only T cells (MST=32 days), peginterferonalfa-2aalone with T cells provided only a minor, but significant, survivaladvantage (MST=35 days; P=0.0276). (E1)-3s plus peginterferonalfa-2awithout T cells did not improve survival significantly.

The antigen density measured for NCI-N87 [247,000(±65,000) Trop-2/cell]and Capan-1 [157,000 (±37,000) Trop-2/cell] was not significantlydifferent. Compared to NCI-N87, Capan-1 cells were >5-fold moresensitive (IC₅₀=2 nM vs. >10 nM) to direct inhibition bypeginterferonalfa-2a in vitro (not shown). (E1)-3s does not cross-reactwith mouse Trop-2 or CD3 (not shown), and NOD-SCID mice are T-celldeficient.

Discussion

This section discusses results presented in Examples 23-26. We describedin Examples 1 and 2 above the use of the (X)-3s bsAb format forredirecting T cell-mediated therapy of both hematopoietic and solidtumors using several example constructs, including (E1)-35, (19)-3s and(20)-3s. In one in vivo experiment from that study, where Capan-1xenografts were treated with (E1)-3s, we included groups withpeginterferonalfa-2a, because prior (unpublished) data showed thatCapan-1 was inhibited by IFN-α. The striking enhancement observed withthe addition of IFN-α spurred further investigation, leading to thecurrent studies. The results of studies with T cell redirectingbispecific antibodies, in combination with peginterferonalfa-2a arereported herein. The studies were extended until all groups reachedtheir MST, confirming that IFN-α can enhance the in-vivo efficacy ofT-cell killing of an IFN-α-sensitive cell line. IFN-α also can enhanceT-cell-mediated killing of a cell line that is weakly sensitive to thedirect actions of IFN-α. These in vivo studies were performed followingmethods, including dosing and schedules, typically used with BiTEconstructs.

Flieger and colleagues demonstrated that in-vitro killing by CD3⁺CD56⁺NK-T cells, which were expanded ex vivo and redirected with an EpCAMxCD3BiTE (MT110), was enhanced with either IFN-α or IL-2 (Flieger et al.,2000, Cancer Immunol Immunother 49:441-8). However, even in the absenceof the bsAb, IFN-α significantly inhibited the target cells. Since acontrol to evaluate potential direct effects of IFN-α on target cellswas lacking, the extent to which the enhanced cytotoxicity was due toIFN-α stimulating NK-T cells, compared to direct inhibition of targetcells, could not be determined. Therefore, we measured the sensitivityto IFN-α for both target cells and included groups withpeginterferonalfa-2a only, both in the presence and absence of pan-Tcells. For Capan-1 tumors, which were more sensitive to IFN-α in vitro,peginterferonalfa-2a improved survival in the absence of T cells, andeven more so in the presence of T cells, indicating that IFN-α acted onboth Capan-1 as well as T cells in this model. In the absence of Tcells, peginterferonalfa-2a did not improve survival of mice bearingNCI-N87 xenografts, which were weakly sensitive to IFN-α in vitro,indicating that the enhancement with IFN-α was due primarily to itsactions on T cells. The mechanism of the observed T-cell enhancement byIFN-α is unclear. The increase in CD69 expression attributed to IFN-αwas moderate, but significant, suggesting that the cytokine maypotentiate T-cell activation induced with the bsAb. Additionally, IFN-αspecifically increased (up to 3-fold) the release of IFN-γ, which isconsidered the chief cytotoxic cytokine produced by cytotoxic T cells,whereas none of the other cytokines measured increased consistently.

Combination therapy with IFN-α and a T-cell-redirecting bsAb has notbeen investigated clinically, or even in animal models. However, IL-2was combined with a F(ab′)₂ fragment of an anti-CD3/EpCAM quadroma in aclinical trial (Kroesen et al., 1997, Cancer Immunol Immunother45:203-6), but treatment was limited due to considerable toxicity mostlikely caused by induction of secondary cytokines, known as CRS orcytokine storm. Systemic administration of IL-2 is known to induce acytokine storm (Panelli et al., 2004, J Transl Med 2:17), and theseverity of adverse events associated with CRS, such as with the TGN1412catastrophic trial, are correlated with IL-2 release (Eastwood et al.,2013, Br J Clin Pharmacol 76:299-315). Although it is not without sideeffects, immunotherapy with IFN-α, which is not produced by T cells, isnot typically associated with cytokine storm.

CRS is a risk associated with immunotherapy using any T-cell directedmAb (e.g., Okt3) or bsAb, including BiTE (Klinger et al., 2012, Blood119:6226-33). However, not all bsAb formats necessarily have the samerisk. Brandl et al. reported cytokine induction with blinatumomab, whereresponse levels of IL-2, IL-6, IFN-γ, and TNF-α were variable amongdonors and typically peaked at >1 ng/mL, with some donors reachinglevels as high as 5 ng/mL (Brandl et al., 2007, Cancer ImmunolImmunother 56:1551-63). We lacked a suitable BiTE, or equivalentconstruct, for direct comparison with (E1)-3s. However, we were able tocompare the relative cytokine-inducing potency between the (X)-3s andBiTE formats, using a CD19×CD3 BiTE (identical sequence as blinatumomab)and (19)-3s made by DNL®. The 19-3 BiTE induced similar cytokine levelsas reported by Brandl and colleagues under similar conditions. Thelevels of the five cytokines measured were 7-13-fold higher for 19-3BiTE, compared to those of (19)-3s. The use of foreign lymphoma cells(Raji) caused a mixed lymphocyte reaction, which increased the baselinecytokine levels, particularly for IL-2. BiTE, but not (19)-3s, increasedthe cytokine levels well above the mixed lymphocyte baseline level.Using NCI-N87 gastric carcinoma cells as the target for (E1)-3s did notincrease baseline cytokine levels. We observed an expected variabilityin donor response to (E1)-3s; however, the resulting cytokine levelswere even lower than those induced by (19)-3s, particularly for TNF-αand IFN-γ, which were <100 pg/mL. Nevertheless, one of five donors hadelevated levels (˜1 ng/mL) of IFN-γ and IL-6. Addition of IFN-α(peginterferonalfa-2a) to (E1)-3s increased IFN-γ 2-3-fold, but did notconsistently affect the levels of the other cytokines. These resultssuggest that compared to other constructs, such as BiTE, the (X)-3s bsAbformat is less likely to induce CRS, and the addition of IFN-α to atherapeutic regimen does not increase this risk.

We observed considerable variability in the potency of donor T cells.The in vitro results shown in FIG. 28 represent the most and leastactive T cells that we have tested, with a 100-fold difference inpotency (IC₅₀=0.37 pM vs. 39 pM) for killing NCI-N87; however, anIC₅₀=1-5 pM is most representative (>10 donors) and the low-activity Tcells was atypical. Notably, lysis with the weaker T cells was augmentedby IFN-α more than with the potent T cells.

EpCAM is a widely exploited TAA that is overexpressed in manycarcinomas. However, the heterogeneous expression of EpCAM in carcinomasand the fact that EpCAM is not tumor-specific, since it is expressed onmost normal epithelia, raise concerns that immunotherapy directedtowards EpCAM could have severe side effects (Balzar et al., 1999, J MolMed (Berl) 77:699-712; Momburg et al., 1987, Cancer Res 47:2883-91).Like EpCAM, Trop-2 is highly expressed in diverse carcinomas, but itsexpression in normal tissues is under debate. Several reports indicatethat, in contrast to tumor cells, somatic adult tissues show little orno Trop-2 expression, which is invariably upregulated in tumors,regardless of baseline expression in normal tissues (Wang et al., 2008,Mol Cancer Ther 7:280-5; Zhang et al., 1997, Science 276:1268-72).However, recent evidence indicates expression of Trop-2 on epithelia ofseveral normal tissues (Trerotola et al., 2013, Oncogene 32:222-33).Nonetheless, expression of Trop-2 in Cynomolgus monkeys did not resultin toxicities after administrations of reasonably high doses of hRS7(humanized anti-Trop-2) conjugated with SN-38 as an antibody-drugconjugate (ADC) (Cardillo et al., 2011, Clin Cancer Res 17:3157-69).Further, in clinical studies with this anti-Trop-2 ADC, no increasednormal organ toxicity other than manageable neutropenia and diarrhea,expected from the drug (a metabolite of irinotecan), was observed attherapeutic doses (Starodub et al., Proceedings of the 105th AnnualMeeting of the American Association for Cancer Research. 2014 (abstrCT206)). Thus, immunotherapy, including T-cell-redirected therapy, usingTrop-2 for tumor targeting, is expected to have a similar, or greater,therapeutic index compared to similar regimens targeting EpCAM.

This is the first report of trogocytosis between target tumor and Tcells mediated by a bsAb. This finding demonstrates that thetarget/T-cell conjugates induced with (E1)-3s have functionalimmunologic synapses. We observed a similar bi-directional trogocytosisbetween B cells and T cells, which was mediated by (19)-3s (unpublisheddata), and believe this is likely a common phenomenon with T-cellredirecting bsAbs.

Example 27 Further Studies with E1-3 Bispecific Antibodies

Summary

A T-cell redirecting bispecific tandem scFv, E1-3, was produced asdescribed in Example 19 above, using the variable domains of hRS7(humanized anti-Trop-2 mAb) and Okt-3 (anti-CD3 mAb). The studiesreported in this Example continue and expand on the results shown inExamples 20-25. Any discrepancies between the instant reported resultsand those shown in Examples 20-25 are based on the collection ofadditional data. T-cell activation, proliferation, cytokine inductionand cytotoxicity were evaluated ex vivo using PBMCs or purified T cellswith human pancreatic (Capan-1 and BxPC-3) and gastric (NCI-N87) cancercell lines as target cells. In vivo activity was assayed with NCI-N87xenografts that were inoculated s.c. in a mixture with twice the numberof human PBMCs and matrigel.

Results

In the presence of target cells and PBMCs, E1-3 potently induced T-cellactivation, proliferation and a dose-dependent cytokine production ofIL-2 (>2 ng/mL), IL-6 (>1 ng/mL), IL-10 (>7 ng/mL), TNF-α (>1 ng/mL) andIFN-γ (>50 ng/mL). Using 3-5 different T cell donors, E1-3 mediated ahighly potent T-cell lysis of BxPC-3 [IC₅₀=0.09(±0.04) pM], Capan-1[IC₅₀=1.2(±1.1) pM] and NCI-N87 [IC₅₀=1.2(±1.2) pM] target cells invitro. In vivo, two 50-μg doses of E1-3 given three days apart cured 6of 8 mice bearing NCI-N87 xenografts (P<0.0001; Log-Rank). Tumors in thecontrol group (PBMCs only) reached the endpoint (TV>1 cm³) with a medianof 39.5 days. Seven of 8 animals had not reached the endpoint, with sixof the mice remaining tumor-free in the E1-3 group when the experimentwas terminated after 176 days.

T-cell activation and proliferation—Purified CD8⁺ T cells were mixed 5:1with NCI-N87 cells, treated for 18 h with 0.01 nM E1-3 and analyzed byflow cytometry. CD69 was upregulated by E1-3 in the presence of targetcells (not shown). Treatments with omission of E1-3 or NCI-N87 targetcells did not induce CD69 expression (not shown). Additionally, T cellsexperienced an increase in forward (FSC) and side scattering (SSC) afterculture in the presence of E1-3 and target cells (not shown). T-cellproliferation was evident after three days (P<0.005, data not shown).

Cytokine release—The ability of E1-3 bispecific tandem scFv to inducerelease of cytokines IFN-γ, TNF-α, IL-2, IL-6 and IL-10 as a function ofdosage was determined. As shown in FIG. 31, the E1-3 bispecific antibodyeffectively induced cytokine release in the picomolar concentrationrange.

In vitro T-cell mediated killing—The ability of E1-3 to induce T-cellmediated killing of target pancreatic and gastric cancer cells wasdetermined in the presence of purified CD8⁺ T cells (1.2×10⁵/well). Anexemplary dose-response curve using T-cells from a representative donorare shown in FIG. 32. In this experiment, the IC₅₀ values for E1-3 were0.6 pM for Capan-1, 0.1 pM for BxPC-3 and 0.3 pM for NCI-N87.

In vivo anti-tumor effects of E1-3—Nude mice bearing NCI-N87 xenograftswere treated with two 50-μg doses of E1-3 given three days apart. Thetreatment (FIG. 33A) cured 6 of 8 mice bearing the human gastric cancerxenografts (P<0.0001; Log-Rank). In comparison with tumors in thecontrol group (treated with PBMCs only) reached the endpoint (TV>1 cm³)with a median of 39.5 days (FIG. 33B). When the study was terminatedafter 176 days, seven of eight animals in the E1-3 group had not reachedthe endpoint.

Conclusions

The studies above show that Trop-2 is an attractive target forT-cell-mediated killing of pancreatic, gastric and other epithelialcancers. The E1-3 anti-Trop-2×anti-CD3 bispecific antibody inducedpotent T-cell activation and cytokine production. E1-3 was highlyeffective at killing solid tumors in vitro and in vivo.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and used without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations may be applied to the COMPOSITIONS and METHODS andin the steps or in the sequence of steps of the METHODS described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A method of inducing an immune response to aTrop-2 expressing cancer comprising: a) administering to a subject witha Trop-2 expressing cancer a bispecific antibody that comprises (i) atleast one anti-Trop-2 antibody or antigen-binding fragment thereof and(ii) one anti-CD3 antibody or antigen-binding fragment thereof; and b)administering to the subject at least one therapeutic agent selectedfrom the group consisting of (iii) interferon-α; and (iv) a checkpointinhibitor antibody that binds to an antigen selected from the groupconsisting of PD1, PD-L1, LAG3, B7-H3, B7-H4, KIR and TIM3.
 2. Themethod of claim 1, wherein the checkpoint inhibitor antibody is selectedfrom the group consisting of pembrolizumab (MK-3475), nivolumab(BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, durvalumab(MEDI4736), atezolizumab (MPDL3280A), BMS-936559, lirilumab, IPH2101,and avelumab.
 3. The method of claim 1, wherein the interferon-α isselected from the group consisting of free interferon, PEGylatedinterferon, an interferon fusion protein or interferon conjugated to anantibody.
 4. The method of claim 1, wherein the bispecific antibodycomprises at least one antibody fragment selected from the groupconsisting of a scFv, a Fab and a dAb.
 5. The method of claim 1, whereinthe bispecific antibody comprises a humanized RS7 antibody orantigen-binding fragment thereof comprising the light chain CDRsequences CDR1 (KASQDVSIAVA, SEQ ID NO:115); CDR2 (SASYRYT, SEQ IDNO:116); and CDR3 (QQHYITPLT, SEQ ID NO:117) and the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:118); CDR2 (WINTYTGEPTYTDDFKG, SEQ IDNO:119) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:120).
 6. The method of claim1, wherein the bispecific antibody comprises an Okt3 antibody orantigen-binding fragment thereof.
 7. The method of claim 1, wherein thebispecific antibody comprises the amino acid sequence of SEQ ID NO:107.